Use of A33 antigens and JAM-IT

ABSTRACT

The present invention relates to compositions and methods of treating and diagnosing disorders characterized the by the presence of antigens associated with inflammatory diseases and/or cancer.

The present application is a divisional of application Ser. No.10/633,008, filed Jul. 31, 2003, now U.S. Pat. No. 7,192,589, which is acontinuation in part of application Ser. No. 10/265,542 filed Oct. 3,2002, now abandoned, which is a continuation in part of PCTinternational application no. PCT/US00/04414, filed Feb. 22, 2000, as acontinuation in part of PCT international application no.PCT/US00/14042, filed May 22, 2000, as a continuation in part of PCTinternational application no. PCT/US00/32678, filed Dec. 1, 2000, as acontinuation in part of U.S. application Ser. No. 09/254,465, filed Mar.5, 1999, now, U.S. Pat. No. 6,410,708, as a continuation in part of PCTinternational application no. PCT/US99/05028, filed Mar. 8, 1999, as acontinuation in part of U.S. application Ser. No. 09/380,138, filed Aug.25, 1999 now abandoned as a continuation in part of U.S. applicationSer. No. 09/380,139, filed Aug. 25, 1999, now abandoned, as acontinuation in part of PCT international application no.PCT/US98/19330, filed Sep. 16, 1998, and as a continuation in part ofU.S. application Ser. No. 09/953,499, filed Sep. 14, 2001, now U.S. Pat.No. 6,838,554, which in turn is a continuation application, claimingpriority under 35 U.S.C. §120 as a continuation of PCT internationalapplication no. PCT/US98/24855, filed Nov. 20, 1998.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the identification, isolationand recombinant production of novel DNA and novel polypeptides thepresence of which is associated with inflammatory diseases (inflammationassociated antigens) and/or cancer, and to compositions and methods forthe diagnosis and treatment of conditions characterized by suchantigens.

2. Description of the Related Art

The inflammatory response is complex and is mediated by a variety ofsignaling molecules produced locally by mast cells, nerve endings,platelets, leukocytes and complement activation. Certain of thesesignaling molecules cause the endothelial cell lining to become moreporous and/or even to express selections which act as cell surfacemolecules which recognize and attract leukocytes through specificcarbohydrate recognition. Stronger leukocyte binding is mediated byintegrins, which mediate leukocyte movement through the endothelium.Additional signaling molecules act as chemoattractants, causing thebound leukocytes to crawl towards the source of the attractant. Othersignaling molecules produced in the course of an inflammatory responseescape into the blood and stimulate the bone marrow to produce moreleukocytes and release them into the blood stream.

Inflammation is typically initiated by an antigen, which can bevirtually any molecule capable of initiating an immune response. Undernormal physiological conditions these are foreign molecules, butmolecules generated by the organism itself can serve as the catalyst asis known to occur in various disease states.

T-cell proliferation in a mixed lymphocyte culture or mixed lymphocytereaction (MLR) is an established indication of the ability of a compoundto stimulate the immune system. In an inflammatory response, theresponding leukocytes can be neutrophilic, eosinophilic, monocytic orlymphocytic. Histological examination of the affected tissues providesevidence of an immune stimulating or inhibiting response. See CurrentProtocols in Immunology, ed. John E. Coligan, 1994, John Wiley and Sons,Inc.

Inflammatory bowel disease (IBD) is a term used to collectively describegut disorders including both ulcerative colitis (UC) and Crohn'sdisease, both of which are classified as distinct disorders, but sharecommon features and likely share pathology. The commonality of thediagnostic criteria can make it difficult to precisely determine whichof the two disorders a patient has; however the type and location of thelesion in each are typically different. UC lesions arecharacteristically a superficial ulcer of the mucosa and appear in thecolon, proximal to the rectum. CD lesions are characteristicallyextensive linear fissures, and can appear anywhere in the bowel,occasionally involving the stomach, esophagus and duodenum.

Conventional treatments for IBD usually involve the administration ofanti-inflammatory or immunosuppressive agents, such as sulfasalazine,corticosteroids, 6-mercaptopurine/azathoprine, or cyclosporine all ofwhich only bring partial relief to the afflicted patient. However whenanti-inflammatory/immunosuppressive therapies fail, colectomies are thelast line of defense. Surgery is required for about 30% of CD patientswithin the first year after diagnosis, with the likelihood for operativeprocedure increasing about 5% annually thereafter. Unfortunately, CDalso has a high rate of reoccurrence as about 5% of patients requiresubsequent surgery after the initial year. UC patients further have asubstantially increased risk of developing colorectal cancer. Presumablythis is due to the recurrent cycles of injury to the epithelium,followed by regrowth, which continually increases the risk of neoplastictransformation.

A recently discovered member of the immunoglobulin superfamily known asJunctional Adhesion Molecule (JAM) has been identified to be selectivelyconcentrated at intercellular junctions of endothelial and epithelialcells of different origins. Martin-Padura, I. et al., J. Cell Biol.142(1): 117-27 (1998). JAM is a type I integral membrane protein withtwo extracellular, intrachain disulfide loops of the V-type. JAM bearssubstantial homology to A33 antigen (FIG. 1 or FIG. 18). A monoclonalantibody directed to JAM was found to inhibit spontaneous andchemokine-induced monocyte transmigration through an endothelial cellmonolayer in vitro. Martin-Padura, supra. It has been recentlydiscovered that JAM expression is increased in the colon of CRF2-4−/−mice with colitis. CRF 2-4−/− (IL-10R subunit knockout mice) develop aspontaneous colitis mediated by lymphocytes, monocytes and neutrophils.Several of the animals also developed colon adenocarcinoma. As a result,it is likely that the polypeptides disclosed herein are expressed inelevated levels in or otherwise associated with human diseases such asinflammatory bowel disease, other inflammatory diseases of the gut aswell as colorectal carcinoma.

JAM and the polypeptides disclosed herein bear significant homology toA33 antigen, a known colorectal cancer-associated marker. The A33antigen is expressed in more than 90% of primary or metastatic coloncancers as well as normal colon epithelium. In carcinomas originatingfrom the colonic mucosa, the A33 antigen is expressed homogeneously inmore than 95% of all cases. The A33 antigen, however, has not beendetected in a wide range of other normal tissues, i.e., its expressionappears to be organ specific. Therefore, the A33 antigen appears to playan important role in the induction of colorectal cancer.

Since colon cancer is a widespread disease, early diagnosis andtreatment is an important medical goal. Diagnosis and treatment of coloncancer can be implemented using monoclonal antibodies (mAbs) specifictherefore having fluorescent, nuclear magnetic or radioactive tags.Radioactive gene, toxins and/or drug tagged mAbs can be used fortreatment in situ with minimal patient description. mAbs can also beused to diagnose during the diagnosis and treatment of colon cancers.For example, when the serum levels of the A33 antigen are elevated in apatient, a drop of the levels after surgery would indicate the tumorresection was successful. On the other hand, a subsequent rise in serumA33 antigen levels after surgery would indicate that metastases of theoriginal tumor may have formed or that new primary tumors may haveappeared.

Such monoclonal antibodies can be used in lieu of, or in conjunctionwith surgery and/or other chemotherapies. For example, preclinicalanalysis and localization studies in patients infected with colorectalcarcinoma with a mAb to A33 are described in Welt et al., J. Clin.Oncol. 8: 1894-1906 (1990) and Welt et al., J. Clin. Oncol. 12:1561-1571 (1994), while U.S. Pat. No. 4,579,827 and U.S. Ser. No.424,991 (E.P. 199,141) are directed to the therapeutic administration ofmonoclonal antibodies, the latter of which relates to the application ofanti-A33 mAb.

SUMMARY OF THE INVENTION

In one aspect, the present invention concerns a method of treating aninflammatory disorder in a mammal, comprising administering to themammal a therapeutically effective amount of an antagonist of a nativesequence STIgMA polypeptide.

In one embodiment, the STIgMA polypeptide is selected from the groupconsisting of polypeptides of SEQ ID NOS: 2, 32, 33, and 34.

In another embodiment, the antagonist is an antibody, such as amonoclonal antibody, which may have non-human complementaritydetermining region (CDR) residues and contains human framework region(FR) residues.

In a further embodiment, the antagonist is an immunoadhesin, whichcomprises a STIgMA extracellular domain sequence fused to animmunoglobulin constant region sequence.

In another embodiment, the inflammatory disorder is selected from thegroup consisting of: inflammatory bowel disease; systemic lupuserythematosus; rheumatoid arthritis; juvenile chronic arthritis;spondyloarthropathies; systemic sclerosis, for example, scleroderma;idiopathic inflammatory myopathies for example, dermatomyositis,polymyositis; Sjögren's syndrome; systemic vaculitis; sarcoidosis;autoimmune hemolytic anemia for example, immune pancytopenia, paroxysmalnocturnal hemoglobinuria; autoimmune thrombocytopenia, for example,idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia;thyroiditis, for example, Grave's disease, Hashimoto's thyroiditis,juvenile lymphocytic thyroiditis, atrophic thyroiditis; diabetesmellitus, immune-mediated renal disease, for example,glomerulonephritis, tubulointerstitial nephritis; demyelinating diseasesof the central and peripheral nervous systems such as multiplesclerosis, idiopathic polyneuropathy; hepatobiliary diseases such asinfectious hepatitis such as hepatitis A, B, C, D, E and othernonhepatotropic viruses; autoimmune chronic active hepatitis; primarybiliary cirrhosis; granulomatous hepatitis; and sclerosing cholangitis;inflammatory and fibrotic lung diseases (e.g., cystic fibrosis);gluten-sensitive enteropathy; Whipple's disease; autoimmune orimmune-mediated skin diseases including bullous skin diseases, erythemamultiforme and contact dermatitis, psoriasis; allergic diseases of thelung such as eosinophilic pneumonia, idiopathic pulmonary fibrosis andhypersensitivity pneumonitis, transplantation associated diseasesincluding graft rejection and graft-versus host disease.

In a different aspect, the invention concerns a method of diagnosing aninflammatory disorder in a mammal, said method comprising detecting thelevel of expression of a gene encoding a STIgMA polypeptide (a) in atest sample of cells obtained from said mammal, and (b) in a controlsample of known normal cells of the same cell type, wherein a higherlevel of expression of said gene in the test sample as compared to thecontrol sample is indicative of the presence of an immune relateddisorder in the mammal from which the test tissue cells were obtained.

In a further aspect, the invention concerns a method of diagnosing aninflammatory disorder in a mammal, said method comprising (a) contactingan anti-STIgMA antibody with a test sample of cells obtained from saidmammal, and (b) detecting the formation of a complex between theantibody and STIgMA polypeptide in the test sample, wherein formation ofsaid complex is indicative of the presence of an inflammatory disorderin said mammal.

The invention further concerns an isolated antibody which specificallybinds a STIgMA polypeptide.

In a different aspect, the invention concerns an isolated nucleic acidmolecule comprising a nucleotide sequence encoding a polypeptide havingat least about 80%, or at least about 85% or at least about 90% or atleast about 95% or at least about 99% sequence identity with the aminoacid sequence of amino acids 21 to 276 of SEQ ID NO: 32, or amino acids21 to 182 of SEQ ID NO: 33, or amino acids 21 to 180 of SEQ ID NO: 34.

The invention further concerns vectors and cells comprising the nucleicacids of the invention.

In another aspect, the invention concerns a polypeptide comprising anamino acid sequence selected from the group consisting of amino acids 21to 276 of SEQ ID NO: 32, amino acids 21 to 182 of SEQ ID NO: 33, andamino acids 21 to 180 of SEQ ID NO: 34.

In yet another aspect, the invention concerns an immunoadhesincomprising amino acids from 1 or about 21 to about 276 of SEQ ID NO: 32,or amino acids from 1 or about 21 to about 182 of SEQ ID NO: 33, oramino acids 1 or about 21 to about 180 of SEQ ID NO: 34, fused to animmunoglobulin constant region sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 show a comparison between the polypeptides encoded by A33 antigen(SEQ ID NO: 6), DNA40628 (SEQ ID NO: 1), DNA45416 (SEQ ID NO: 2),DNA35638 (SEQ ID NO: 9) and JAM (SEQ ID NO: 10).

FIG. 2 shows the derived amino acid sequence (SEQ ID NO: 1) of a nativesequence PRO301 polypeptide. This polypeptide is 299 amino acids long,having signal sequence at residue 1 to 27, an extracellular domain atresidue 28 to about 235, Ig superfamily homology at residue 94 to 235, apotential transmembrane domain at residue 236 to about 258, and anintracellular domain at about residue 259 to 299.

FIG. 3 shows the amino acid sequence (SEQ ID NO: 2) derived fromnucleotides 119-1081 of the nucleotide sequence shown in FIGS. 6A and 6B(DNA45416, SEQ ID NO: 7). Also shown in FIG. 3 as underlines are thelocations of a glycosoaminoglycan site and a transmembrane domain.

FIG. 4A shows the consensus assembly DNA35936 (SEQ ID NO: 3), and FIG.4B shows consen01 (SEQ ID NO: 4) which were both used in the isolationof DNA40628 (SEQ ID NO: 11). FIG. 4C shows consen02 (DNA42257) (SEQ IDNO: 5) which was used in the isolation of DNA45416 (SEQ ID NO: 7).

FIG. 5 shows the nucleotide sequence of a native sequence DNA40628 cDNA(SEQ ID NO: 11), which is a native sequence PRO301 cDNA also designatedas “UNQ264” and/or “DNA40628-1216”.

FIGS. 6A & B show a nucleotide sequence DNA45416 (SEQ ID NO: 7) which isa native sequence PRO362 cDNA also designated as “UNQ317” and/or“DNA45416-1251”. Also presented are the initiator methionine and theprotein translation for a full-length PRO362 polypeptide (SEQ ID NO: 2).

FIG. 7 shows the nucleotide sequence (SEQ ID NO: 8) of a native sequencePRO245 cDNA, wherein the nucleotide sequence is designated as “UNQ219”and/or “DNA35638”.

FIG. 8 shows the oligonucleotide sequences OLI2162 (35936.f1) (SEQ IDNO: 12), OLI2163 (35936.p1) (SEQ ID NO: 13), OLI2164 (35936.f2) (SEQ IDNO: 14), OLI2165 (35936.r1) (SEQ ID NO: 15), OLI2166 (35936.f3) (SEQ IDNO: 16), OLI2167 (35936.r2) (SEQ ID NO: 17) which were used in theisolation of DNA40628.

FIGS. 9A & B show a double stranded representation of the DNA42257(consen02) (SEQ ID NO: 5) along with the locations of fiveoligonucleotide primers, showed in underline, all used in the isolationof DNA45416 (SEQ ID NO: 7). The oligonucleotides depicted are: 42257.f1(SEQ ID NO: 18), 42257.f2 (SEQ ID NO: 19), 42257.r1 (SEQ ID NO: 20),42257.r2 (SEQ ID NO: 21) and 42257.p1 (SEQ ID NO: 22).

FIGS. 10A and B describe the Blast score, match and percent homologyalignment between 2 overlapping fragments of DNA40628 and A33_HUMAN, ahuman A33 antigen precursor. FIG. 10A compares the coded residues 24 to283 of DNA40628 (SEQ ID NO: 23) with the coded residues 17 to 284 ofA33_HUMAN (SEQ ID NO: 24); FIG. 10B compares the coded residues 21 to239 of DNA40628 (SEQ ID NO: 25) with the coded residues 12 to 284 ofA33_HUMAN (SEQ ID NO: 26), respectively.

FIG. 11 shows the derived amino acid sequence of a native sequencePRO245 polypeptide (SEQ ID NO: 9) encoded by the nucleotide sequence ofFIG. 7 (DNA35638, SEQ ID NO: 8). This polypeptide is a 312 amino acidsin length, having signal sequence at residue 1 to 28 and a potentialtransmembrane domain at about residue 237 to about 259.

FIG. 12 indicates a 25.3% identity between the amino acid sequenceencoded by DNA40628 (SEQ ID NO: 1) and A33 antigen (SEQ ID NO: 6).

FIG. 13 indicates a 20.8% identity between the amino acid sequenceencoded by DNA45416 (SEQ ID NO: 2) and A33 antigen (SEQ ID NO: 6).

FIG. 14 indicates a 24.3% identity between the amino acid sequenceencoded by DNA35638 (SEQ ID NO: 9) and A33 antigen (SEQ ID NO: 6).

FIG. 15 indicates a 67.6% identity between the amino acid sequenceencoded by DNA40628 (SEQ ID NO: 1) and JAM (SEQ ID NO: 10).

FIG. 16 indicates a 23.3% identity between the amino acid sequenceencoded by DNA45416 (SEQ ID NO: 2) and JAM (SEQ ID NO: 10).

FIG. 17 indicates a 34.2% identity between the amino acid sequenceencoded by DNA35638 (SEQ ID NO: 29) and JAM (SEQ ID NO: 10).

FIG. 18 indicates a 26% identity between the amino acid sequence encodedby A33 antigen (SEQ ID NO: 6) and JAM (SEQ ID NO: 10).

FIG. 19 shows the results of the dot blot hybridization proceduredescribed in Example 8.

FIG. 20 shows the results of the TAQMAN™ mRNA expression assay describedin Example 9.

FIG. 21 shows the binding of protein encoded by DNA40628 to humanneutrophils as described in Example 7.

FIG. 22 shows the amino acid sequence (SEQ ID NO: 31) of PRO1868 with ▾,representing a putative signal cleavage site, ●, representing conservedextracellular cysteines, the transmembrane domain underlined and theoverlying dotted lines, representing potential N-glycosylation sites.This polypeptide is 310 amino acids in length, having signal sequence atresidue 1 to 30 and a potential transmembrane domain at about residue242 to about 266.

FIG. 23 shows in situ hybridization of PRO362 in mouse liver frozensections.

FIG. 24 shows in situ hybridization of PRO362 in human liver frozensections.

FIG. 25 shows in situ hybridization of PRO362 in colon macrophages (FIG.25A), Kupffer cells (FIG. 25B), adrenal macrophages (FIG. 25C), Hofbauercells (FIG. 25D).

FIG. 26 shows in situ hybridization of PRO362 mRNA in Synovial cells.

FIG. 27 shows in situ hybridization of PRO362 RNA in type A synovialcells.

FIG. 28 shows in situ hybridization of PRO362 mRNA in brain microgliacells.

FIG. 29 shows in situ hybridization of PRO362 mRNA in cells from humanasthmatic tissue.

FIG. 30 shows in situ hybridization of PRO362 mRNA in cells from humanchronic hepatitis tissue.

FIG. 31 shows in situ hybridization of PRO245 mRNA in lymph node andtonsil high endothelial venule (HEV) cells of normal human tissue.

FIG. 32 shows in situ hybridization of PRO245 mRNA in arteriolarendothelium of inflamed and normal human lung tissue, as well as innormal seminiferous tubules of testis in spermatogenic cells.

FIG. 33 shows in situ hybridization of PRO245 mRNA in human testicular,lung and mammary carcinoma tissue.

FIG. 34 shows in situ hybridization of PRO245 mRNA in human breastcarcinoma tissue.

FIG. 35 shows immunohistochemical analysis of PRO362 in macrophages.

FIG. 36 shows immunohistochemical analysis of PRO362 in Kupffer cells.

FIG. 37 shows immunohistochemical analysis of PRO362 in microglialcells.

FIG. 38 shows immunohistochemical analysis of PRO362 in Hofbauer cells.

FIG. 39 shows SDS-PAGE analysis of PRO1868 mRNA detected byreverse-transcriptase PCR(RT-PCR) in T cell lines J45 and Molt4, and Bcell lines JY, RPMI8866 and RAMOS.

FIG. 40 shows a schematic summarizing PRO245 binding by cytolytic Tcells, NK-T cells, and NK cells.

FIG. 41 shows flow cytometry results of binding between NK (CD56+) cellsand PRO245-Fc fusion protein.

FIG. 42 shows flow cytometry results of binding between peripheral blooddendritic cells (PBDCs) and PRO245-Fc fusion protein.

FIG. 43 shows a graph representing flow cytometry results of bindingbetween J45 T cells and PRO245-Fc fusion protein.

FIG. 44 shows flow cytometry results of binding between J45 T cells andPRO245-Fc fusion protein.

FIG. 45 shows a graph of flow cytometry results that demonstrates theability of excess His-tagged-PRO1868 to block J45 cell adherence toPRO245-Fc fusion protein.

FIG. 46 shows flow cytometry results of the ability ofHis-tagged-PRO1868 to block PRO245-Fc fusion protein to NK (CD56+)cells.

FIG. 47 shows a graph representing the percent adhesion of labeled J45cells to wells coated with varying concentrations of PRO245.

FIG. 48 shows immunoprecipitation of biotinylated J45 cells toFc-cross-linked PRO245-Fc fusion protein A matrix.

FIG. 49 shows immunoprecipitation of PRO1868 from J45 and PBMC cellsusing PRO245-Fc fusion protein cross-linked protein A matrices.

FIG. 50 shows a graph representing the binding of biotinylated PRO245 towells coated with PRO1868.

FIG. 51 shows a graph representing the binding of biotinylated PRO1868to wells coated with PRO245-Fc.

FIG. 52 shows data representing inhibition of the adhesion of J45 cellsto PRO245-Fc fusion protein by anti-PRO1868 antibodies. Data arerepresentative of three independent experiments; error bars representthe SD in an n=6 condition.

FIG. 53 shows flow cytometry results indicating the ability of 6×His-tagged PRO1868 protein to compete with binding between CD56+ NKcells and PRO245-Fc fusion protein.

FIG. 54 shows binding of PRO1868 to PRO245 expressing CHO cells undervarious conditions.

FIG. 55 shows specific binding of anti-PRO1868 antibodies toPRO245-expressing CHO cells (CuL8r).

FIG. 56 shows the amino acid sequence of human STIgMA (hSTIgMA; SEQ IDNO: 32) and human STIgMA short (hSTIgMA short; SEQ ID NO: 33) andalignment with murine STIgMA (SEQ ID NO: 34). The hydrophobic leadersequence, transmembrane region, and potential N-linked glycosylationsites are shown. The Ig domain boundaries, deduced from the exon-intronboundaries of the human STIgMA gene, are indicated.

FIG. 57. Northern blot analysis showing expression of human STIgMA inplacenta, lung, heart, liver and adrenal gland (A). Two transcripts of1.5 and 1.8 kb were present in the human tissues expressing STIgMA.

FIG. 58. (A) TAQMAN™ PCR analysis showing increased expression of humanSTIgMA in myelomonocytic cell lines HL60 and THP-1 and in differentiatedmacrophages. Low levels of expression were found in Jurkat T cells,MOLT3, MOLT4 and RAMOS B-cell lines. (B) Increased expression of STIgMAmRNA during in vitro monocyte differentiation. Monocytes isolated fromhuman peripheral blood were differentiated by adhering to plastic over 7day period. Total RNA was extracted at different time points duringdifferentiation. (C) Increased expression of STIgMA protein duringmonocyte to macrophage differentiation. Monocytes were treated asindicated in (B), whole cell lysates were run on a gel and transferredto nitrocellulose membrane that was incubated with a polyclonal antibody(4F7) to human STIgMA. The polyclonal antibody recognized a 48 and 38kDa band possibly representing the long and the short form of STIgMA.

FIG. 59. Molecular characterization of huSTIgMA protein in cell lines.(A) HuSTIgMA-gd was transiently expressed in 293E cells,immunoprecipitated with anti gd and blots incubated with anti gd or apolyclonal antibody to the extracellular domain of STIgMA. (B) huSTIgMAexpressed in 293 cells is a monomeric N-glycosylated protein. STIgMA istyrosine phosphorylated upon treatment of HEK293 cells with sodiumpervanadate but does not recruit Syk kinase. Phosphorylated STIgMAmigrated at a slightly higher molecular mass compared tonon-phosphorylated STIgMA.

FIG. 60. Selective expression of STIgMA on human monocyte-derivedmacrophages. Peripheral blood mononuclear cells were stained withantibodies specific for B, T, NK cells, monocytes and with a ALEXA™ A488conjugated monoclonal antibody (3C9) to STIgMA. Expression was absent inall peripheral blood leukocytes as well as in monocyte derived dendriticcells, but was expressed in in vitro differentiated macrophages.

FIG. 61. STIgMA mRNA and protein expression was increased by IL-10 anddexamethasone. (A) Real-time PCR shows increased expression of STIgMAmRNA following treatment with IL-10, TGFbeta and was highly induced bydexamethasone but was down-regulated by treatment with LPS, IFNγ, andTNFα. (B) Ficoll-separated peripheral blood mononuclear cells weretreated with various cytokines and dexamethasone for 5 days anddouble-stained with anti CD14 and anti STIgMA. Flow analysis showed adramatic increase in STIgMA expression on the surface of monocytestreated with dexamethasone and after treatment with IL-10 and LPS.

FIG. 62. Subcellular localization of STIgMA in monocyte-derivedmacrophages. Monocytes were cultured for 7 days in macrophagedifferentiation medium, fixed in acetone and stained with polyclonalanti STIgMA antibody 6F1 or CD63 and secondary goat-anti rabbit FITC.Cells were studied in a confocal microscope. STIgMA is found in thecytoplasm were it co-localizes with the lysosomal membrane protein CD63.STIgMA was also expressed at the trailing and leading edges ofmacrophages in a pattern similar to that of F-actin. Scale bar=10 μm.

FIG. 63. Localization of STIgMA mRNA in chronic inflammatory diseases.In situ hybridization showed the presence of STIgMA mRNA in alveolarmacrophages obtained from tissue of a patient with pneumonia (A, B) or apatient with chronic asthma (C, D). STIgMA mRNA was also expressed inliver Kupffer cells in tissue obtained from a liver biopsy of a patientwith chronic hepatitis (E, F).

FIG. 64. STIgMA mRNA expression was increased in inflamed synovium.STIGMA mRNA was low or absent in synovial membranes of a joint obtainedfrom a knee replacement of a patient with no joint inflammation (A, C)but was highly expressed in cells, potentially synoviocytes or synovialmacrophages, in the pannus of a patient with osteoarthritis (B, D).

FIG. 65. Detection of STIgMA protein with polyclonal antibody 6F1 incells lining the synovium of a patient with degenerative joint disease(A, B, C). No immunohistochemical detection of STIgMA was found in acontrol synovium (D).

FIG. 66. STIgMA protein was expressed in a subtype of tissue residentmacrophages and its expression was increased in chronic inflammatorydiseases. (A) STIgMA was expressed on the membrane of CHO cells stablyexpressing STIgMA. High expression of STIgMA protein was found inalveolar macrophages (B) in tissues obtained from a patient with chronicasthma. (C) Expression of STIgMA in histiocytes of the human smallintestine. The section was obtained from surgically removed tissue andcould have contained a neoplasm. (D) Expression of STIgMA protein inHofbauer cells in human pre-term placenta. High expression of STIgMAprotein in macrophages was present in the adrenal gland (E) and inKupffer cells of human liver (F). Staining was performed on 5 μm thickacetone-fixed sections using DAB as the chromogen. Images werephotographed at a 20× and 40× magnification.

FIG. 67. Immunohistochemical staining of CD68 and STIgMA on a vascularplaque obtained from a patient with atherosclerosis. Consecutivesections were fixed and stained with a monoclonal antibody to human CD68(A, B) and a polyclonal antibody 6F1 raised against human STIgMA (C, D).STIgMA appeared in a population of macrophages and phoam cells presentin the atherosclerotic plaque, and overlapped with CD68 positivemacrophages, as judged from staining on consecutive sections.Magnification: 10× (A, C) and 20× (B, D).

FIG. 68. Co-staining of STIgMA and CD68 on heart interstitialmacrophages. 5 μm sections were obtained from a human heart (autopsy)and stained with a monoclonal antibody to STIgMA (3C9) and a secondaryanti-mouse FITC-labeled antibody. CD68 was detected by staining with aPE-labeled monoclonal antibody to CD68. Magnification: 20×.

FIG. 69. STIgMA mRNA is significantly increased in colon tissue obtainedfrom patients with ulcerative colitis, Crohn's disease, chronicocclusive pulmonary disease (COPD) and asthma. Real-time PCR wasperformed on total RNA extracted from the various tissues. mRNA forSTIgMA was significantly increased in tissues obtained from patientswith ulcerative colitis, Crohn's disease and COPD. Statistical analysiswas performed using the Mann-Whitney U-test.

FIG. 70. Cells expressing human STIgMA showed increased adherence tohuman endothelial cells. (A) STIgMA was stably expressed in a humanJurkat T-cell line. (B) Cells were preloaded with the fluorescent dyeBCECF (Molecular Probes, Oregon) and added to a 96 well plate coatedwith a monolayer of human umbilical vein endothelial cells (HUVEC)treated with or without 10 ng/ml TNFα. After 3 washes, fluorescence wascounted in a spectro-fluorometer which indicated the number of cellsthat remain adherent to the HUVEC cells. The graph was representative of4 independent experiments.

FIG. 71. Inhibition of progression of collagen-induced arthritis (CIA)mouse model by muSTIgMA IgG-Fc fusion protein. A group of (CIA) mice(n=7) was given 100 μg of muSTIgMA IgG-Fc fusion protein (squares),whereas a CIA mouse control group (n=8) received 100 μg of murine IgG1(circles), 3 times per week for 6 weeks. Mice were examined daily forsigns of inflammation and scored on a scale of 0-16 (details in Example25) and the results were plotted graphically (mean±SD, Student's T testp-value=0.0004 for control IgG1 vs. test muSTIgMA protein).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT I. Definitions

The terms “PRO301”, “PRO362, “PRO245”, “PRO1868” or “PRO301polypeptide,” “PRO362 polypeptide,” “PRO245 polypeptide,” “PRO1868” and“cancer associated antigen” when used herein encompass native sequencePRO301, PRO362, PRO245, or PRO1868 respectively and variants thereof(which are further defined herein). In addition the terms “PRO301” and“JAM-1” are used interchangeably, as are the terms “PRO362,” “JAM4,”STIGMA,” and “STIgMA.” Further, the terms “PRO245,” “JAM-IT” and “JAM-2”are used interchangeably, as are the terms “PRO1868,” “SHATR” and“JAM-3.” The PRO301, PRO362, PRO245 or PRO1868 polypeptides may beisolated from a variety of sources, such as from human tissue types orfrom another source, or prepared by recombinant or synthetic methods. Asnoted, the listed designations are used to refer to the respectivenative sequence molecules and their variants.

Thus, for example, STIgMA includes a polypeptide comprising amino acids1 to 321 of SEQ ID NO: 2; amino acids 1 to X of SEQ ID NO: 2 (wherein Xis any of amino acids 271 to 280); amino acids 21 to 321 of SEQ ID NO:2; amino acids 21 to X of SEQ ID NO: 2 (wherein X is any of amino acids271 to 280); amino acids 1 to 399 of SEQ ID NO: 32; amino acids 21 to399 of SEQ ID NO: 32; amino acids 1 to 305 of SEQ ID NO: 33; amino acids21 to 305 of SEQ ID NO: 33; amino acids 1 to 280 of SEQ ID NO: 34; aminoacids 21 to 280 of SEQ ID NO: 34; the extracellular domains and variantsin which part or all of the transmembrane domain has been deleted orinactivated.

The term “inflammatory disease” and “inflammatory disorder” are usedinterchangeably and mean a disease or disorder in which a component ofthe immune system of a mammal causes, mediates or otherwise contributesto an inflammatory response contributing to morbidity in the mammal.Also included are diseases in which reduction of the inflammatoryresponse has an ameliorative effect on progression of the disease.Included within this term are immune-mediated inflammatory diseases,including autoimmune diseases.

The term “T-cell mediated” disease means a disease in which T cellsdirectly or indirectly mediate or otherwise contribute to morbidity in amammal. The T cell mediated disease may be associated with cell mediatedeffects, lymphokine mediated effects, etc. and even effects associatedwith B cells if the B cells are stimulated, for example, by thelymphokines secreted by T cells.

Examples of immune-related and inflammatory diseases, some of which areT cell mediated, include, without limitation, inflammatory boweldisease, systemic lupus erythematosus, rheumatoid arthritis, juvenilechronic arthritis, spondyloarthropathies, systemic sclerosis(scleroderma), idiopathic inflammatory myopathies (dermatomyositis,polymyositis), Sjögren's syndrome, systemic vaculitis, sarcoidosis,autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnalhemoglobinuria), autoimmune thrombocytopenia (idiopathicthrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis(Grave's disease, Hashimoto's thyroiditis, juvenile lymphocyticthyroiditis, atrophic thyroiditis), diabetes mellitus, immune-mediatedrenal disease (glomerulonephritis, tubulointerstitial nephritis),demyelinating diseases of the central and peripheral nervous systemssuch as multiple sclerosis, idiopathic polyneuropathy, hepatobiliarydiseases such as infectious hepatitis (hepatitis A, B, C, D, E and othernonhepatotropic viruses), autoimmune chronic active hepatitis, primarybiliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis,inflammatory and fibrotic lung diseases (e.g., cystic fibrosis),gluten-sensitive enteropathy, Whipple's disease, autoimmune orimmune-mediated skin diseases including bullous skin diseases, erythemamultiforme and contact dermatitis, psoriasis, allergic diseases of thelung such as eosinophilic pneumonia, idiopathic pulmonary fibrosis andhypersensitivity pneumonitis, transplantation associated diseasesincluding graft rejection and graft-versus host disease.

“Tumor”, as used herein, refers to all neoplastic cell growth andproliferation whether malignant or benign, and all pre-cancerous cellsand tissues.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include breast cancer, prostatecancer, colon cancer, squamous cell cancer, small-cell lung cancer,non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, colorectal cancer, endometrial carcinoma, salivarygland carcinoma, kidney cancer, liver cancer, vulval cancer, thyroidcancer, hepatic carcinoma and various types of head and neck cancer.

“Treatment” is an intervention performed with the intention ofpreventing the development or altering the pathology of a disorder.Accordingly, “treatment” refers to both therapeutic treatment andprophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those in which thedisorder is to be prevented. In treatment of an immune related disease,a therapeutic agent may directly alter the magnitude of response of acomponent of the immune response, or render the disease more susceptibleto treatment by other therapeutic agents, e.g., antibiotics,antifungals, anti-inflammatory agents, chemotherapeutics, etc.

The “pathology” of an immune related disease includes all phenomena thatcompromise the well-being of the patient. This includes, withoutlimitation, abnormal or uncontrollable cell growth (neutrophilic,eosinophilic, monocytic, lymphocytic cells), antibody production,auto-antibody production, complement production, interference with thenormal functioning of neighboring cells, release of cytokines or othersecretory products at abnormal levels, suppression or aggravation of anyinflammatory or immunological response, infiltration of inflammatorycells (neutrophilic, eosinophilic, monocytic, lymphocytic) into cellularspaces, etc.

The term “mammal” as used herein refers to any animal classified as amammal, including, without limitation, humans, domestic and farmanimals, and zoo, sports or pet animals such horses, pigs, cattle, dogs,cats and ferrets, etc. In a preferred embodiment of the invention, themammal is a human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. I¹³¹,I¹²⁵, Y⁹⁰ and Re¹⁸⁶), chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant or animalorigin, or fragments thereof.

A “chemotherapeutic agent” is a compound useful in the treatment ofcancer. Examples of chemotherapeutic agents include adriamycin,doxorubicin, epirubicin, 5-fluorouracil, cytosine arabinoside (“Ara-C”),cyclophosphamide, thiotepa, busulfan, cytoxin, taxoids, e.g. paclitaxel(Taxol®, Bristol-Myers Squibb Oncology, Princeton, N.J.) and doxetaxel(Taxotere®, Rhône-Poulenc Roher, Antony, France), toxotere,methotrexate, cisplatin, melphalan, vinblastine, bleomycin, etoposide,ifosfamide, mitomycin C, mitoxantrone, vincristine (Loucristine),vinorelbine, carboplatin, teniposide, daunomycin, caminomycin,aminopterin, dactinomycin, mitomycins, esperamicins (see U.S. Pat. No.4,675,187), melphalan and other related nitrogen mustards. Also includedin this definition are hormonal agents that act to regulate or inhibithormonal action on tumors such as tamoxifen and onapristone.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell, especially cancer cellsexpressing or overexpressing any of the genes identified herein, eitherin vitro or in vivo. Thus, the growth inhibitory agent is one whichsignificantly reduces the percentage of cells expressing oroverexpressing such genes in S phase. Examples of growth inhibitoryagents include agents that block cell cycle progression (at a placeother than S phase), such as agents that induce G1 arrest and M-phasearrest. Classical M-phase blockers include the vinca alkaloids(vincristine and vinblastine), taxol, and topo II inhibitors such asdoxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Thoseagents that arrest G1 also spill over into S-phase arrest, for example,DNA alkylating agents such tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in The Molecular Basis of Cancer,Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation,oncogenes, and antineoplastic drugs” by Murakami et al. (WB Saunders,Philadelphia, 1995), especially page 13.

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone, parathyroid hormone, thyroxine, insulin,proinsulin, relaxin, prorelaxin, glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH), hepatic growth factor, fibroblast growthfactor, prolactin, placental lactogen, tumor necrosis factor-α and β,mullerian-inhibiting substance, mouse gonadotropin-associated peptide,inhibin, activin, vascular endothelial growth factor, integrin,thrombopoietin (TPO), nerve growth factors such as NGF-β,platelet-growth factor, transforming growth factors (TGFs) such as TGF-αand TGF-β, insulin-like growth factor-I and -II, erythropoietin (EPO),osteoinductive factors, interferons such as interferon-α, -β, and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF),granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF),interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-11, IL-12, a tumor necrosis factor such as TNF-α orTNF-β, and other polypeptide factors including LIF and kit ligand (KL).As used herein, the term cytokine includes proteins from natural sourcesor from recombinant cell culture and biologically active equivalents ofthe native sequence cytokines.

“Therapeutically effective amount” is the amount of active PRO301,PRO362, PRO245 or PRO1868 antagonist or agonist which is required toachieve a measurable inhibition or stimulation, as the case may be, ofthe inflammatory response.

A “native sequence PRO301, PRO362, PRO245 or PRO1868”, comprises apolypeptide having the same amino acid sequence as a PRO301, PRO362,PRO245 or PRO1868 respectively, derived from nature. Such nativesequence PRO301, PRO362, PRO245 or PRO1868 can be isolated from natureor can be produced by recombinant or synthetic means. The terms “nativesequence PRO301”, “native sequence PRO362,” “native sequence PRO245” and“native sequence PRO1868” specifically encompass naturally-occurringtruncated or secreted forms of PRO301, PRO362, PRO245 and PRO1868respectively (e.g., an extracellular domain sequence),naturally-occurring variant forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants of PRO301, PRO362, PRO245 orPRO1868, respectively.

In one embodiment, the native sequence PRO301 is a mature or full-lengthnative sequence PRO301 comprising amino acids 1 to 299 of FIG. 2 (SEQ IDNO: 1), with or without the N-terminal signal sequence, with or withoutthe initiating methionine at position 1, with or without the potentialtransmembrane domain at about position 236 to about 258, and with orwithout the intracellular domain at about position 259 to 299.

In another embodiment, the native sequence STIgMA polypeptide is amature or full-length native sequence PRO362 comprising amino acids 1 to321 of FIG. 3 (SEQ ID NO: 2), with or without an N-terminal signalsequence, with or without the initiating methionine at position 1, withor without of any or all of the potential transmembrane domain, at aboutpositions 276-306, and with or without the intracellular domain at aboutpositions 307 to 321. In a further embodiment, the native sequenceSTIgMA polypeptide is a mature or full-length polypeptide comprisingamino acids 1 to 399 of SEQ ID NO: 32 (huSTIgMA), with or without anN-terminal signal sequence, with or without the initiating methionine atposition 1, and with or without of any or all of the transmembranedomain at about positions 277 to 300. In a still further embodiment, thenative sequence STIgMA polypeptide is a mature or full-lengthpolypeptide comprising amino acids 1 to 305 of SEQ ID NO: 33 (huSTIgMAshort), with or without an N-terminal signal sequence, with or withoutthe initiating methionine at position 1, and with or without of any orall of the transmembrane domain at about positions 183 to 206. In adifferent embodiment, the native sequence STIgMA polypeptide is a matureor full length polypeptide comprising amino acids 1 to 280 of SEQ ID NO:34 (muSTIgMA), with or without an N-terminal signal sequence, with orwithout the initiating methionine at positions 1, and with or without ofany or all of the transmembrane domain at about positions 181 to 204.

In yet another embodiment, the native sequence PRO245 polypeptide is amature or full-length native sequence PRO245 polypeptide comprisingamino acids 1 to 312 of FIG. 11 (SEQ ID NO: 9), with or without anN-terminal signal sequence, with or without the initiating methionine atposition 1, with or without a potential transmembrane domain, and withor without an intracellular domain.

In yet another embodiment, the native sequence PRO1868 polypeptide is amature or full-length native sequence PRO1868 polypeptide comprisingamino acids 1 to 310 of FIG. 22 (SEQ ID NO: 31), with or without theN-terminal signal sequence at about positions 1 to 30, with or withoutthe initiating methionine at position 1, with or without the potentialtransmembrane domain at about position 242 to about 266, and with orwithout the intracellular domain at about position 267 to 310.

The “PRO301, PRO362 (STIgMA), PRO245 or PRO1868 extracellular domain” or“PRO301, PRO362, PRO245 or PRO1868 ECD” refers to a form of the PRO301,PRO362 (STIgMA), PRO245 or PRO1868 polypeptide, respectively, which isessentially free of the transmembrane and cytoplasmic domains of therespective full length molecules. Ordinarily, PRO301 ECD, PRO362(STIgMA) ECD, PRO245 ECD or PRO1868 ECD will have less than 1% of suchtransmembrane and/or cytoplasmic domains and preferably, will have lessthan 0.5% of such domains.

Optionally, PRO301 polypeptide ECD will comprise amino acid residues 1or about 28 to X, wherein X is any amino acid from amino acid 231 toamino acid 241 of FIG. 2 (SEQ ID NO: 1).

Optionally, PRO362 (STIgMA) polypeptide ECD will comprise amino acidresidues 1 or about 21 to X of FIG. 3 (SEQ ID NO: 2), or SEQ ID NO: 32,where X is any amino acid from about 271 to 281, or amino acid residues1 or about 21 to X of SEQ ID NO: 33, where X is any amino acid fromabout 178 to 186, or amino acid residues 1 or about 21 to X of SEQ IDNO: 34, wherein X is any amino acid from about 176 to 184 of SEQ ID NO:34.

Optionally, PRO245 polypeptide ECD will comprise amino acid residues 1or about 29 to X, wherein X is any amino acid from amino acid 232 toamino acid 242.

Optionally, PRO1868 polypeptide ECD will comprise amino acid residues 1or about 31 to X, wherein X is any amino acid from amino acid 237 toamino acid 247.

It will be understood that any transmembrane domain identified for thePRO301, PRO362 (STIgMA), PRO245 or PRO1868 polypeptides of the presentinvention is identified pursuant to criteria routinely employed in theart for identifying that type of hydrophobic domain. The exactboundaries of a transmembrane domain may vary but most likely by no morethan about 5 amino acids at either end of the domain as initiallyidentified.

“PRO301 variant” means an active PRO301 as defined below having at leastabout 80% amino acid sequence identity to (a) a DNA molecule encoding aPRO301 polypeptide, with or without its native signal sequence, with orwithout the initiating methionine, with or without the potentialtransmembrane domain, and with or without the intracellular domain or(b) the complement of the DNA molecule of (a). In a particularembodiment, the PRO301 variant has at least about 80% amino acidsequence homology with the PRO301 having the deduced amino acid sequenceshown in FIG. 1 (SEQ ID NO: 1) for a full-length native sequence PRO301.Such PRO301 variants include, for instance, PRO301 polypeptides whereinone or more amino acid residues are added, or deleted, at the N- orC-terminus of the sequence of FIG. 2 (SEQ ID NO: 1). Preferably, thenucleic acid or amino acid sequence identity is at least about 85%, morepreferably at least about 90%, and even more preferably at least about95%. Preferably, the highest degree of sequence identity occurs withinthe extracellular domains (amino acids 28 to 235 of FIG. 2, SEQ ID NO:1).

“PRO245 variant” means an active PRO245 as defined below having at leastabout 80% amino acid sequence identity to (a) a DNA molecule encoding aPRO245 polypeptide, with or without its native signal sequence, with orwithout the initiating methionine, with or without the potentialtransmembrane domain, and with or without the intracellular domain or(b) the complement of the DNA molecule of (a). In a particularembodiment, the PRO245 variant has at least about 80% amino acidsequence homology with the PRO245 having the deduced amino acid sequenceshown in FIG. 1 (SEQ ID NO: 9) for a full-length native sequence PRO245.Such PRO245 variants include, for instance, PRO245 polypeptides whereinone or more amino acid residues are added, or deleted, at the N- orC-terminus of the sequence of SEQ ID NO: 9. Preferably, the nucleic acidor amino acid sequence identity is at least about 85%, more preferablyat least about 90%, and even more preferably at least about 95%.

“PRO362 variant” means an active PRO362 polypeptide as defined belowhaving at least about 80% amino acid sequence identity to (a) a DNAmolecule encoding a PRO362 polypeptide, with or without its nativesignal sequence, with or without the initiating methionine, with orwithout the potential transmembrane domain, and with or without theintracellular domain or (b) the complement of the DNA molecule of (a).In a particular embodiment, the PRO362 variant has at least about 80%amino acid sequence homology with the PRO362 polypeptide having thededuced amino acid sequence shown in FIG. 3 (SEQ ID NO: 2) for afull-length native sequence PRO362 polypeptide. Such PRO362 polypeptidevariants include, for instance, PRO362 polypeptides wherein one or moreamino acid residues are added, or deleted, at the N- or C-terminus ofthe sequence of FIG. 3 (SEQ ID NO: 2). Ordinarily, a PRO362 polypeptidevariant will have at least about 80% amino acid sequence identity,preferably at least about 85% amino acid sequence identity, morepreferably at least about 90% amino acid sequence identity and even morepreferably at least about 95% amino acid sequence identity with theamino acid sequence of FIG. 3 (SEQ ID NO: 2). Preferably, the highestdegree of sequence identity occurs within the extracellular domains(amino acids 1 to X of FIG. 3, SEQ ID NO: 2, where X is any amino acidresidue from 271 to 281).

A “STIgMA variant” specifically includes the PRO362 variants definedabove, along with variants of SEQ ID NOS: 32, 33, and 34. In particular,STIgMA variants specifically include an active STIgMA polypeptide asdefined below having at least about 80% amino acid sequence identity to(a) a DNA molecule encoding a polypeptide or SEQ ID NO: 32, 33, or 34,with or without its native signal sequence, with or without theinitiating methionine, with or without all or part of the potentialtransmembrane domain, and with or without the intracellular domain or(b) the complement of the DNA molecule of (a). In a particularembodiment, the STIgMA variant has at least about 80% amino acidsequence homology with the STIgMA polypeptide having the deduced aminoacid sequence of SEQ ID NO: 32, 33, or 34. Such STIgMA variants include,for instance, STIgMA polypeptides wherein one or more amino acidresidues are added, or deleted, at the N- or C-terminus of the sequenceof SEQ ID NOS: 32, 33, and 34. Ordinarily, a STIgMA polypeptide variantwill have at least about 80% amino acid sequence identity, preferably atleast about 85% amino acid sequence identity, more preferably at leastabout 90% amino acid sequence identity and even more preferably at leastabout 95% amino acid sequence identity with the amino acid sequence ofSEQ ID NO: 32, 33, or 34. Preferably, the highest degree of sequenceidentity occurs within the extracellular domains.

“PRO1868 variant” means an active PRO1868 polypeptide as defined belowhaving at least about 80% amino acid sequence identity to (a) a DNAmolecule encoding a PRO1868 polypeptide, with or without its nativesignal sequence, with or without the initiating methionine, with orwithout the potential transmembrane domain, and with or without theintracellular domain or (b) the complement of the DNA molecule of (a).In a particular embodiment, the PRO1868 variant has at least about 80%amino acid sequence homology with the with the PRO1868 polypeptidehaving the deduced amino acid sequence of SEQ ID NO: 31 encoding afull-length native sequence PRO1868 polypeptide. Such PRO1868polypeptide variants include, for instance, PRO1868 polypeptides whereinone or more amino acid residues are added, or deleted, at the N- orC-terminus of the sequence of SEQ ID NO: 31. Ordinarily, a PRO1868polypeptide variant will have at least about 80% amino acid sequenceidentity, preferably at least about 85% amino acid sequence identity,more preferably at least about 90% amino acid sequence identity and evenmore preferably at least about 95% amino acid sequence identity with theamino acid sequence of SEQ ID NO: 31.

“Percent (%) amino acid sequence identity” with respect to the PRO301,PRO362 (STIgMA), PRO245 or PRO1868 sequences identified herein isdefined as the percentage of amino acid residues in a candidate sequencethat are identical with the amino acid residues in the PRO301, PRO362(STIgMA), PRO245 or PRO1868 sequence, respectively, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for measuring alignment, including any algorithmsneeded to achieve maximal alignment over the full length of thesequences being compared.

“Percent (%) nucleic acid sequence identity” with respect to thePRO301-, PRO362 (STIgMA), PRO245- or PRO1868-encoding sequencesidentified herein (e.g., DNA40628, DNA45416, DNA35638, DNA77624) isdefined as the percentage of nucleotides in a candidate sequence thatare identical with the nucleotides in the PRO301-, PRO362 (STIgMA)-,PRO245- or PRO1868-encoding sequence, respectively, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity. Alignment for purposes of determining percentnucleic acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor measuring alignment, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe nucleic acid. An isolated nucleic acid molecule is other than in theform or setting in which it is found in nature. Isolated nucleic acidmolecules therefore are distinguished from the nucleic acid molecule asit exists in natural cells. However, an isolated nucleic acid moleculeincludes nucleic acid molecules contained in cells that ordinarilyexpress an encoded polypeptide where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

An “isolated” PRO301, PRO362(STIgMA), PRO245 or PRO1868polypeptide-encoding nucleic acid molecule is a nucleic acid moleculethat is identified and separated from at least one contaminant nucleicacid molecule with which it is ordinarily associated in the naturalsource of the PRO301, PRO362 (STIgMA), PRO245 or PRO1868polypeptide-encoding nucleic acid. An isolated PRO301, PRO362 (STIgMA),PRO245 or PRO1868 polypeptide-encoding nucleic acid molecule is otherthan in the form or setting in which it is found in nature. IsolatedPRO301, PRO362, PRO245 or PRO1868 polypeptide-encoding nucleic acidmolecules therefore are distinguished from the DNA40628, DNA45416,DNA35638 or DNA77624 nucleic acid molecules, respectively, as theyexists in natural cells. However, an isolated PRO301, PRO362, PRO245 orPRO1868 polypeptide-encoding nucleic acid molecule includes PRO301,PRO362, PRO245 or PRO1868 polypeptide-encoding nucleic acid moleculescontained in cells that ordinarily express PRO301, PRO362, PRO245 orPRO1868 polypeptide where, for example, the nucleic acid molecule is ina chromosomal location different from that of natural cells.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The term “antibody” is used in the broadest sense and specificallycovers, without limitation, single anti-PRO301, anti-PRO362(anti-STIgMA), anti-PRO245 or anti-PRO1868 monoclonal antibodies(including agonist, antagonist, and neutralizing antibodies) andanti-PRO301, anti-PRO362, anti-PRO245 or anti-PRO1868 antibodycompositions with polyepitopic specificity. The term “monoclonalantibody” as used herein refers to an antibody obtained from apopulation of substantially homogeneous antibodies, i.e., the individualantibodies comprising the population are identical except for possiblenaturally-occurring mutations that may be present in minor amounts.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature that can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, may be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising a polypeptide of the invention fused to a “tagpolypeptide”. The tag polypeptide has enough residues to provide anepitope against which an antibody can be made, yet is short enough suchthat it does not interfere with activity of the polypeptide to which itis fused. The tag polypeptide preferably also is fairly unique so thatthe antibody does not substantially cross-react with other epitopes.Suitable tag polypeptides generally have at least six amino acidresidues and usually between about 8 and 50 amino acid residues(preferably, between about 10 and 20 amino acid residues).

“Active” or “activity” in the context of variants of the polypeptide ofthe invention refers to form(s) of proteins of the invention whichretain the biologic and/or immunologic activities of a native ornaturally-occurring polypeptide of the invention.

“Biological activity” in the context of an antibody, polypeptide oranother molecule that can be identified by the screening assaysdisclosed herein (e.g. an organic or inorganic small molecule, peptide,etc.) refers, in part, to the ability of such molecules to alterinfiltration of inflammatory cells into a tissue, to alter T-cellproliferation and to alter lymphokine release by cells. Anotherpreferred activity an affect on vascular permeability.

The term “antagonist” is used in the broadest sense, and includes anymolecule that partially or fully blocks, inhibits, or neutralizes abiological activity of a native polypeptide of the invention disclosedherein. In a similar manner, the term “agonist” is used in the broadestsense and includes any molecule that mimics or stimulates a biologicalactivity of a native polypeptide of the invention disclosed herein.Suitable agonist or antagonist molecules specifically include agonist orantagonist antibodies or antibody fragments, fragments or amino acidsequence variants of native polypeptides of the invention, peptides,small molecules, including small organic molecules, etc.

A “small molecule” is defined herein to have a molecular weight belowabout 600 daltons.

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingthe same structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.The term “antibody” is used in the broadest sense and specificallycovers, without limitation, intact monoclonal antibodies, polyclonalantibodies, multispecific antibodies (e.g. bispecific antibodies) formedfrom at least two intact antibodies, and antibody fragments so long asthey exhibit the desired biological activity.

“Native antibodies” and “native immunoglobulins” are usuallyheterotetrameric glycoproteins of about 150,000 daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is linked to a heavy chain by one covalent disulfide bond,while the number of disulfide linkages varies among the heavy chains ofdifferent immunoglobulin isotypes. Each heavy and light chain also hasregularly spaced intrachain disulfide bridges. Each heavy chain has atone end a variable domain (V_(H)) followed by a number of constantdomains. Each light chain has a variable domain at one end (V_(L)) and aconstant domain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light and heavy chain variable domains.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (CDRs) orhypervariable regions both in the light-chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR). The variable domains of native heavy andlight chains each comprise four FR regions, largely adopting abeta-sheet configuration, connected by three CDRs, which form loopsconnecting, and in some cases forming part of, the beta-sheet structure.The CDRs in each chain are held together in close proximity by the FRregions and, with the CDRs from the other chain, contribute to theformation of the antigen-binding site of antibodies (see Kabat et al.,NIH Publ. No. 91-3242, Vol. I, pages 647-669 (1991)). The constantdomains are not involved directly in binding an antibody to an antigen,but exhibit various effector functions, such as participation of theantibody in antibody-dependent cellular toxicity.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng.8(10):1057-1062 [1995]); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment. The designation “Fc” reflects theability to crystallize readily. Pepsin treatment yields an F(ab′)₂fragment that has two antigen-combining sites and is still capable ofcross-linking antigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chainconstant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 [1975], or may be made byrecombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The“monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352:624-628 [1991] and Marks et al., J. Mol. Biol., 222: 581-597 (1991),for example. See also U.S. Pat. Nos. 5,750,373, 5,571,698, 5,403,484 and5,223,409 which describe the preparation of antibodies using phagemidand phage vectors.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 [1984]).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies are human immunoglobulins(recipient antibody) in which several or all residues from acomplementarity-determining region (CDR) of the recipient are replacedby residues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity, andcapacity. In some instances, certain Fv framework region (FR) residuesof the human immunoglobulin can also be replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues which are found neither in the recipient antibody nor in theimported CDR or framework sequences. These modifications are made tofurther refine and maximize antibody performance. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin sequence. The humanized antibody optimally also willcomprise at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. For further details, see Joneset al., Nature, 321: 522-525 (1986); Reichmann et al., Nature, 332:323-329 [1988]; and Presta, Curr. Op. Struct. Biol., 2: 593-596 (1992).The humanized antibody includes a “primatized” antibody where theantigen-binding region of the antibody is derived from an antibodyproduced by immunizing macaque monkeys with the antigen of interest.Antibodies containing residues from Old World monkeys are also possiblewithin the invention. See, for example, U.S. Pat. Nos. 5,658,570;5,693,780; 5,681,722; 5,750,105; and 5,756,096.

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thesFv to form the desired structure for antigen binding. For a review ofsFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315(1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

An “isolated” polypeptide, including an isolated antibody, is one whichhas been identified and separated and/or recovered from a component ofits natural environment. Contaminant components of its naturalenvironment are materials which would interfere with diagnostic ortherapeutic uses for the antibody, and may include enzymes, hormones,and other proteinaceous or nonproteinaceous solutes. In preferredembodiments, the polypeptide will be purified (1) to greater than 95% byweight of the compound as determined by the Lowry method, and mostpreferably more than 99% by weight, (2) to a degree sufficient to obtainat least 15 residues of N-terminal or internal amino acid sequence byuse of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGEunder reducing or nonreducing conditions using Coomassie blue or,preferably, silver stain. Isolated compound, e.g. antibody or otherpolypeptide, includes the compound in situ within recombinant cellssince at least one component of the compound's natural environment willnot be present. Ordinarily, however, isolated compound will be preparedby at least one purification step.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to a compound,e.g. antibody or polypeptide, so as to generate a “labeled” compound.The label may be detectable by itself (e.g. radioisotope labels orfluorescent labels) or, in the case of an enzymatic label, may catalyzechemical alteration of a substrate compound or composition which isdetectable.

By “solid phase” is meant a non-aqueous matrix to which the compound ofthe present invention can adhere. Examples of solid phases encompassedherein include those formed partially or entirely of glass (e.g.,controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g., an affinity chromatography column). This term alsoincludes a discontinuous solid phase of discrete particles, such asthose described in U.S. Pat. No. 4,275,149.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as the anti-ErbB2 antibodies disclosed herein and, optionally, achemotherapeutic agent) to a mammal. The components of the liposome arecommonly arranged in a bilayer formation, similar to the lipidarrangement of biological membranes.

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

II. Compositions and Methods of the Invention

A. Preparation of the PRO301, PRO362, PRO245 or PRO1868 Polypeptides

1. Full-Length PRO301, PRO362, PRO245 or PRO1868 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO301, PRO362, PRO245 or PRO1868. In particular, Applicants haveidentified and isolated cDNA encoding a PRO301, PRO362, PRO245 orPRO1868 polypeptide, as disclosed in further detail in the Examplesbelow. Using BLAST and FastA sequence alignment computer programs,Applicants found that full-length native sequence PRO301 (FIG. 2, SEQ IDNO: 1), PRO362 (FIG. 3, SEQ ID NO: 3), PRO245 (FIG. 11, SEQ ID NO: 9)and PRO1868 (SEQ ID NO: 31) have significant homology to both A33antigen and JAM. (See FIGS. 1, 12-18). Accordingly, it is presentlybelieved that PRO301, PRO362, PRO245 and PRO1868 disclosed in thepresent application are newly identified members of the A33 antigenprotein family and may be associated with inflammatory disorders such asinflammatory bowel disease as well as human neoplastic diseases such ascolorectal cancer.

2. PRO301, PRO362, PRO245 or PRO1868 Variants

In addition to the full-length native sequence PRO301, PRO362, PRO245 orPRO1868 described herein, it is contemplated that PRO301, PRO362, PRO245or PRO1868 variants can be prepared. PRO301, PRO362, PRO245 or PRO1868variants can be prepared by introducing appropriate nucleotide changesinto the PRO301, PRO362, PRO245 or PRO1868 DNA, respectively, or bysynthesis of the desired PRO301, PRO362, PRO245 or PRO1868 polypeptides.Those skilled in the art will appreciate that amino acid changes mayalter post-translational processes of the PRO301, PRO362, PRO245 orPRO1868, such as changing the number or position of glycosylation sitesor altering the membrane anchoring characteristics.

Variations in the native full-length sequence PRO301, PRO362, PRO245 orPRO1868 or in various domains of the PRO301, PRO362, PRO245 or PRO1868described herein, can be made, for example, using any of the techniquesand guidelines for conservative and non-conservative mutations setforth, for instance, in U.S. Pat. No. 5,364,934. Variations may be asubstitution, deletion or insertion of one or more codons encoding thePRO301, PRO362, PRO245 or PRO1868 that results in a change in the aminoacid sequence of the PRO301, PRO362, PRO245 or PRO1868 as compared withthe native sequence PRO301, PRO362, PRO245 or PRO1868. Optionally thevariation is by substitution of at least one amino acid with any otheramino acid in one or more of the domains of the PRO301, PRO362, PRO245or PRO1868. Guidance in determining which amino acid residue may beinserted, substituted or deleted without adversely affecting the desiredactivity may be found by comparing the sequence of the PRO301, PRO362,PRO245 or PRO1868 with that of homologous known protein molecules andminimizing the number of amino acid sequence changes made in regions ofhigh homology. Amino acid substitutions can be the result of replacingone amino acid with another amino acid having similar structural and/orchemical properties, such as the replacement of a leucine with a serine,i.e., conservative amino acid replacements. Insertions or deletions mayoptionally be in the range of 1 to 5 amino acids. The variation allowedmay be determined by systematically making insertions, deletions orsubstitutions of amino acids in the sequence and testing the resultingvariants for activity in the in vitro assay described in the Examplesbelow.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res, 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the PRO301, PRO362, PRO245 or PRO1868variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids that may be varied along a contiguous sequence. Amongthe preferred scanning amino acids are relatively small, neutral aminoacids. Such amino acids include alanine, glycine, serine, and cysteine.Alanine is typically a preferred scanning amino acid among this groupbecause it eliminates the side-chain beyond the beta-carbon and is lesslikely to alter the main-chain conformation of the variant. Alanine isalso typically preferred because it is the most common amino acid.Further, it is frequently found in both buried and exposed positions[Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol.Biol., 150:1 (1976)]. If alanine substitution does not yield adequateamounts of variant, an isoteric amino acid can be used.

3. Modifications of PRO301, PRO362, PRO245 or PRO1868

Covalent modifications of PRO301, PRO362, PRO245 or PRO1868 are includedwithin the scope of this invention. One type of covalent modificationincludes reacting targeted amino acid residues of the PRO301, PRO362,PRO245 or PRO1868 with an organic derivatizing agent that is capable ofreacting with selected side chains or the N- or C-terminal residues ofthe PRO301, PRO362, PRO245 or PRO1868. Derivatization with bifunctionalagents is useful, for instance, for crosslinking PRO301, PRO362, PRO245or PRO1868 to a water-insoluble support matrix or surface for use in themethod for purifying anti-PRO301, anti-PRO362, anti-PRO245 oranti-PRO1868 antibodies, respectively, and vice-versa. Commonly usedcrosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),bifunctional maleimides such as bis-N-maleimido-1,8-octane and agentssuch as methyl-3-[(p-azidophenyl)dithio]pro-pioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the PRO301, PRO362, PRO245 orPRO1868 polypeptide included within the scope of this inventioncomprises altering the native glycosylation pattern of the polypeptide.“Altering the native glycosylation pattern” is intended for purposesherein to mean deleting one or more carbohydrate moieties found innative sequence PRO301, PRO362, PRO245 or PRO1868, and/or adding one ormore glycosylation sites that are not present in the native sequencePRO301, PRO362, PRO245 or PRO1868, and/or alteration of the ratio and/orcomposition of the sugar residues attached to the glycosylation site(s).

Addition of glycosylation sites to the PRO301, PRO362, PRO245 or PRO1868polypeptide may be accomplished by altering the amino acid sequence. Thealteration may be made, for example, by the addition of, or substitutionby, one or more serine or threonine residues to the native sequencePRO301, PRO362, PRO245 or PRO1868 (for O-linked glycosylation sites).The PRO301, PRO362, PRO245 or PRO1868 amino acid sequence may optionallybe altered through changes at the DNA level, particularly by mutatingthe DNA encoding the PRO301, PRO362, PRO245 or PRO1868 polypeptide atpreselected bases such that codons are generated that will translateinto the desired amino acids.

Another means of increasing the number of carbohydrate moieties on thePRO301, PRO362, PRO245 or PRO1868 polypeptide is by chemical orenzymatic coupling of glycosides to the polypeptide. Such methods aredescribed in the art, e.g., in WO 87/05330 published 11 Sep. 1987, andin Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the PRO301, PRO362, PRO245or PRO1868 polypeptide may be accomplished chemically or enzymaticallyor by mutational substitution of codons encoding for amino acid residuesthat serve as targets for glycosylation. Chemical deglycosylationtechniques are known in the art and described, for instance, byHakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987) and by Edgeet al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage ofcarbohydrate moieties on polypeptides can be achieved by the use of avariety of endo- and exo-glycosidases as described by Thotakura et al.,Meth. Enzymol., 138:350 (1987).

Another type of covalent modification of PRO301, PRO362, PRO245 orPRO1868 comprises linking the PRO301, PRO362, PRO245 or PRO1868polypeptide to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, forexample in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689;4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The PRO301, PRO362, PRO245 or PRO1868 of the present invention may alsobe modified in a way to form a chimeric molecule comprising PRO301,PRO362, PRO245 or PRO1868 fused to another, heterologous polypeptide oramino acid sequence. In one embodiment, such a chimeric moleculecomprises a fusion of the PRO301, PRO362, PRO245 or PRO1868 with a tagpolypeptide which provides an epitope to which an anti-tag antibody canselectively bind. The epitope tag is generally placed at the amino- orcarboxyl-terminus of the PRO301, PRO362, PRO245 or PRO1868. The presenceof such epitope-tagged forms of the PRO301, PRO362, PRO245 or PRO1868can be detected using an antibody against the tag polypeptide. Also,provision of the epitope tag enables the PRO301, PRO362, PRO245 orPRO1868 to be readily purified by affinity purification using ananti-tag antibody or another type of affinity matrix that binds to theepitope tag. In an alternative embodiment, the chimeric molecule maycomprise a fusion of the PRO301, PRO362, PRO245 or PRO1868 with animmunoglobulin or a particular region of an immunoglobulin. For abivalent form of the chimeric molecule, such a fusion could be to the Fcregion of an IgG molecule.

Various tag polypeptides and their respective antibodies are well knownin the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192-194 (1992)]; an α-tubulin epitope peptide [Skinneret al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)].

4. Production and Isolation of PRO301, PRO362 PRO245 or PRO1868

The description below relates primarily to production of PRO301, PRO362,PRO245 or PRO1868 by culturing cells transformed or transfected with avector containing PRO301, PRO362, PRO245 or PRO1868 nucleic acid. It is,of course, contemplated that alternative methods, which are well knownin the art, may be employed to prepare PRO301, PRO362, PRO245 orPRO1868. For instance, the PRO301, PRO362, PRO245 or PRO1868 sequence,or portions thereof, may be produced by direct peptide synthesis usingsolid-phase techniques [see, e.g., Stewart et al., Solid-Phase PeptideSynthesis, W.H. Freeman Co., San Francisco, Calif. (1969); Merrifield,J. Am. Chem. Soc., 85:2149-2154 (1963)]. In vitro protein synthesis maybe performed using manual techniques or by automation. Automatedsynthesis may be accomplished, for instance, using an Applied BiosystemsPeptide Synthesizer (Foster City, Calif.) using manufacturer'sinstructions. Various portions of the PRO301, PRO362, PRO245 or PRO1868may be chemically synthesized separately and combined using chemical orenzymatic methods to produce the full-length PRO301, PRO362, PRO245 orPRO1868.

a. Isolation of DNA Encoding PRO301, PRO362, PRO245 or PRO1868

DNA encoding PRO301, PRO362, PRO245 or PRO1868 may be obtained from acDNA library prepared from tissue believed to possess the PRO301,PRO362, PRO245 or PRO1868 mRNA and to express it at a detectable level.Accordingly, human PRO301, PRO362, PRO245 or PRO1868 DNA can beconveniently obtained from a cDNA library prepared from human tissue,such as described in the Examples. The PRO301-, PRO362-, PRO245- orPRO1868-encoding gene may also be obtained from a genomic library or byoligonucleotide synthesis.

Libraries can be screened with probes (such as antibodies to PRO301,PRO362, PRO245 or PRO1868 or oligonucleotides of at least about 20-80bases) designed to identify the gene of interest or the protein encodedby it. Screening the cDNA or genomic library with the selected probe maybe conducted using standard procedures, such as described in Sambrook etal., Molecular Cloning: A Laboratory Manual (New York: Cold SpringHarbor Laboratory Press, 1989). An alternative means to isolate the geneencoding PRO301, PRO362, PRO245 or PRO1868 is to use PCR methodology[Sambrook et al., supra; Dieffenbach et al., PCR Primer: A LaboratoryManual (Cold Spring Harbor Laboratory Press, 1995)].

The Examples below describe techniques for screening a cDNA library. Theoligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The oligonucleotide is preferably labeled such that it can be detectedupon hybridization to DNA in the library being screened. Methods oflabeling are well known in the art, and include the use of radiolabelslike ³²P-labeled ATP, biotinylation or enzyme labeling. Hybridizationconditions, including moderate stringency and high stringency, areprovided in Sambrook et al., supra.

Sequences identified in such library screening methods can be comparedand aligned to other known sequences deposited and available in publicdatabases such as GenBank or other private sequence databases. Sequenceidentity (at either the amino acid or nucleotide level) within definedregions of the molecule or across the full-length sequence can bedetermined through sequence alignment using computer software programssuch as BLAST, BLAST-2, ALIGN, DNAstar, and INHERIT which employ variousalgorithms to measure homology.

Nucleic acid having protein coding sequence may be obtained by screeningselected cDNA or genomic libraries using the deduced amino acid sequencedisclosed herein for the first time, and, if necessary, usingconventional primer extension procedures as described in Sambrook etal., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

b. Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for PRO301, PRO362, PRO245 or PRO1868production and cultured in conventional nutrient media modified asappropriate for inducing promoters, selecting transformants, oramplifying the genes encoding the desired sequences. The cultureconditions, such as media, temperature, pH and the like, can be selectedby the skilled artisan without undue experimentation. In general,principles, protocols, and practical techniques for maximizing theproductivity of cell cultures can be found in Mammalian CellBiotechnology: A Practical Approach, M. Butler, ed. (M Press, 1991) andSambrook et al., supra.

Methods of transfection are known to the ordinarily skilled artisan, forexample, CaPO4 and electroporation. Depending on the host cell used,transformation is performed using standard techniques appropriate tosuch cells. The calcium treatment employing calcium chloride, asdescribed in Sambrook et al., supra, or electroporation is generallyused for prokaryotes or other cells that contain substantial cell-wallbarriers. Infection with Agrobacterium tumefaciens is used fortransformation of certain plant cells, as described by Shaw et al.,Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) can be employed. General aspects of mammalian cell host systemtransformations have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5772 (ATCC53,635).

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for PRO301-,PRO362-, PRO245- or PRO1868-encoding vectors. Saccharomyces cerevisiaeis a commonly used lower eukaryotic host microorganism.

Suitable host cells for the expression of glycosylated PRO301, PRO362,PRO245 or PRO1868 are derived from multicellular organisms. Examples ofinvertebrate cells include insect cells such as Drosophila S2 andSpodoptera Sf9, as well as plant cells. Examples of useful mammalianhost cell lines include Chinese hamster ovary (CHO) and COS cells. Morespecific examples include monkey kidney CV1 cells transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney cells (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. GenVirol., 36:59 (1977)); Chinese hamster ovary cells/−DHFR(CHO, Urlaub andChasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells(TM4, Mather, Biol. Reprod., 23:243-251 (1980)); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mouse mammarytumor cells (MMT 060562, ATCC CCL51). The selection of the appropriatehost cell is deemed to be within the skill in the art.

c. Selection and Use of a Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding PRO301, PRO362,PRO245 or PRO1868 may be inserted into a replicable vector for cloning(amplification of the DNA) or for expression. Various vectors arepublicly available. The vector may, for example, be in the form of aplasmid, cosmid, viral particle, or phage. The appropriate nucleic acidsequence may be inserted into the vector by a variety of procedures. Ingeneral, DNA is inserted into an appropriate restriction endonucleasesite(s) using techniques known in the art. Vector components generallyinclude, but are not limited to, one or more of a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence. Construction ofsuitable vectors containing one or more of these components employsstandard ligation techniques which are known to the skilled artisan.

The PRO301, PRO362, PRO245 or PRO1868 may be produced recombinantly notonly directly, but also as a fusion polypeptide with a heterologouspolypeptide, which may be a signal sequence or other polypeptide havinga specific cleavage site at the N-terminus of the mature protein orpolypeptide. In general, the signal sequence may be a component of thevector, or it may be a part of the PRO301, PRO362, PRO245 or PRO1868 DNAthat is inserted into the vector. The signal sequence may be aprokaryotic signal sequence selected, for example, from the group of thealkaline phosphatase, penicillinase, 1pp, or heat-stable enterotoxin IIleaders. For yeast secretion the signal sequence may be, e.g., the yeastinvertase leader, alpha factor leader (including Saccharomyces andKluyveromyces″—factor leaders, the latter described in U.S. Pat. No.5,010,182), or acid phosphatase leader, the C. albicans glucoamylaseleader (EP 362,179 published 4 Apr. 1990), or the signal described in WO90/13646 published 15 Nov. 1990. In mammalian cell expression, mammaliansignal sequences may be used to direct secretion of the protein, such assignal sequences from secreted polypeptides of the same or relatedspecies, as well as viral secretory leaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2: plasmid origin is suitable for yeast, andvarious viral origins (SV40, polyoma, adenovirus, VSV or BPV) are usefulfor cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up the PRO301,PRO362, PRO245 or PRO1868 nucleic acid, such as DHFR or thymidinekinase. An appropriate host cell when wild-type DHFR is employed is theCHO cell line deficient in DHFR activity, prepared and propagated asdescribed by Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980).A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979);Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157(1980)]. The trp1 gene provides a selection marker for a mutant strainof yeast lacking the ability to grow in tryptophan, for example, ATCCNo. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)].

Expression and cloning vectors usually contain a promoter operablylinked to the PRO301, PRO362, PRO245 or PRO1868 nucleic acid sequence todirect mRNA synthesis. Promoters recognized by a variety of potentialhost cells are well known. Promoters suitable for use with prokaryotichosts include the β-lactamase and lactose promoter systems [Chang etal., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)],alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel,Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters suchas the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25(1983)]. Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encodingPRO301, PRO362, PRO245 or PRO1868.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

PRO301, PRO362, PRO245 or PRO1868 transcription from vectors inmammalian host cells is controlled, for example, by promoters obtainedfrom the genomes of viruses such as polyoma virus, fowlpox virus (UK2,211,504 published 5 Jul. 1989), adenovirus (such as Adenovirus 2),bovine papilloma virus, avian sarcoma virus, cytomegalovirus, aretrovirus, hepatitis-B virus and Simian Virus 40 (SV40), fromheterologous mammalian promoters, e.g., the actin promoter or animmunoglobulin promoter, and from heat-shock promoters, provided suchpromoters are compatible with the host cell systems.

Transcription of a DNA encoding the PRO301, PRO362, PRO245 or PRO1868 byhigher eukaryotes may be increased by inserting an enhancer sequenceinto the vector. Enhancers are cis-acting elements of DNA, usually aboutfrom 10 to 300 bp, that act on a promoter to increase its transcription.Many enhancer sequences are now known from mammalian genes (globin,elastase, albumin, α-fetoprotein, and insulin). Typically, however, onewill use an enhancer from a eukaryotic cell virus. Examples include theSV40 enhancer on the late side of the replication origin (bp 100-270),the cytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to thePRO301, PRO362, PRO245 or PRO1868 coding sequence, but is preferablylocated at a site 5′ from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding PRO301, PRO362, PRO245 or PRO1868.

Still other methods, vectors, and host cells suitable for adaptation tothe synthesis of PRO301, PRO362, PRO245 or PRO1868 in recombinantvertebrate cell culture are described in Gething et al., Nature,293:620-625 (1981); Mantei et al., Nature, 281:40-46 (1979); EP 117,060;and EP 117,058.

d. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequencePRO301, PRO362, PRO245 or PRO1868 polypeptide or against a syntheticpeptide based on the DNA sequences provided herein or against exogenoussequence fused to PRO301, PRO362, PRO245 or PRO1868 DNA and encoding aspecific antibody epitope.

e. Purification of Polypeptide

Forms of PRO301, PRO362, PRO245 or PRO1868 may be recovered from culturemedium or from host cell lysates. If membrane-bound, it can be releasedfrom the membrane using a suitable detergent solution (e.g. Triton-X100) or by enzymatic cleavage. Cells employed in expression of PRO301,PRO362, PRO245 or PRO1868 can be disrupted by various physical orchemical means, such as freeze-thaw cycling, sonication, mechanicaldisruption, or cell lysing agents.

It may be desired to purify PRO301, PRO362, PRO245 or PRO1868 fromrecombinant cell proteins or polypeptides. The following procedures areexemplary of suitable purification procedures: by fractionation on anion-exchange column; ethanol precipitation; reverse phase HPLC;chromatography on silica or on a cation-exchange resin such as DEAE;chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gelfiltration using, for example, Sephadex G-75; protein A Sepharosecolumns to remove contaminants such as IgG; and metal chelating columnsto bind epitope-tagged forms of the PRO301, PRO362, PRO245 or PRO1868.Various methods of protein purification may be employed and such methodsare known in the art and described for example in Deutscher, Methods inEnzymology, 182 (1990); Scopes, Protein Purification: Principles andPractice, Springer-Verlag, New York (1982). The purification step(s)selected will depend, for example, on the nature of the productionprocess used and the particular PRO301, PRO362, PRO245 or PRO1868produced.

f. Detection of Cell Interactions

To determine whether the polypeptides of the invention are traffickingor cell adhesion molecules, a number of in vitro assays may beperformed.

1) Flow Cytometry/FACS Analysis

To examine the interaction between PRO301, PRO362, PRO245 or PRO1868with specific cell types, biotinylated human IgG fusion proteins, suchas PRO301-human IgG fusion, PRO362-human IgG fusion, PRO245-human IgGfusion or PRO1868-human IgG fusion, may be generated. Cells thatinteract with the biotinylated fusion proteins may be isolated usingstreptavidin-conjugated magnetic beads. The cells that interact with thebiotinylated fusion proteins may be further characterized and analyzedfor surface CD-Ag expression by flow cytometry and/or FACS sorting.Cells examined for interaction with biotinylated PRO301-human IgGfusion, PRO362-human IgG fusion, PRO245-human IgG fusion orPRO1868-human IgG fusion, may include, for example, peripheral bloodcells, such as NK cells, NK/T cells or cytolytic T cells and morespecifically, purified B cells, neutrophils, monocytes or dendriticcells.

The inhibition of the interaction between PRO301, PRO362, PRO245 orPRO1868 with specific cell types may further be characterized byinhibition analysis, specifically the ability of antibodies, such asanti-PRO301, anti-PRO245, anti-PRO362 or anti-PRO1868 to inhibit suchcell interaction.

2) Coimmunoprecipitation

Upon the identification of PRO301, PRO362, PRO245 or PRO1868-interactingcells, further analysis may be performed to identify the particularreceptor responsible for the PRO301, PRO362, PRO245 or PRO1868interaction. For example, coimmunoprecipitation analysis may beperformed to identify the receptor on PRO245-interacting cells.Antibodies against PRO245 may be incubated with the PRO245-interactingcells. The immunoprecipitates may then be analyzed by SDS-PAGE andimmunoblotting with antibodies against potential receptors. To determinewhether the receptor for PRO245 is a protein that belongs to the JAMfamily of proteins, antibodies used for the immunoblotting may includeanti-PRO301, anti-PRO362 or anti-PRO1868. Such analysis may result inthe identification of a pair of interacting proteins that belong to theA33/JAM family of adhesion molecules.

5. Tissue Distribution

The location of tissues expressing the polypeptides of the invention canbe identified by determining, for example, mRNA expression or proteinexpression in various human tissues. The location of such genes providesinformation about which tissues are most likely to be affected by thestimulating and inhibiting activities of the polypeptides of theinvention. The location of a gene in a specific tissue also providessample tissue for the activity blocking assays discussed below.

Gene expression in various tissues may be measured by conventionalSouthern blotting, Northern blotting to quantitate the transcription ofmRNA (Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 [1980]), dotblotting (DNA analysis), or in situ hybridization, using anappropriately labeled probe, based on the sequences provided herein.Alternatively, antibodies may be employed that can recognize specificduplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybridduplexes or DNA-protein duplexes.

Gene expression in various tissues, alternatively, may be measured byimmunological methods, such as immunohistochemical staining of tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequenceof a polypeptide of the invention or against a synthetic peptide basedon the DNA sequences encoding the polypeptide of the invention oragainst an exogenous sequence fused to a DNA encoding a polypeptide ofthe invention and encoding a specific antibody epitope. Generaltechniques for generating antibodies, and special protocols for Northernblotting and in situ hybridization are provided below.

6. Antibody Binding Studies

The activity of the polypeptides of the invention can be furtherverified by antibody binding studies, in which the ability ofanti-PRO301, anti-PRO362, anti-PRO245 or anti-PRO1868 antibodies toinhibit the effect of the PRO301, PRO362, PRO245 or PRO1868 polypeptideson tissue cells is tested. Exemplary antibodies include polyclonal,monoclonal, humanized, bispecific, and heteroconjugate antibodies, thepreparation of which will be described herein below.

Antibody binding studies may be carried out in any known assay method,such as competitive binding assays, direct and indirect sandwich assays,and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual ofTechniques, pp. 147-158 (CRC Press, Inc., 1987).

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample analyte for binding with a limited amountof antibody. The amount of target protein in the test sample isinversely proportional to the amount of standard that becomes bound tothe antibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies preferably are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insolublethree-part complex. See, e.g., U.S. Pat. No. 4,376,110. The secondantibody may itself be labeled with a detectable moiety (direct sandwichassays) or may be measured using an anti-immunoglobulin antibody that islabeled with a detectable moiety (indirect sandwich assay). For example,one type of sandwich assay is an ELISA assay, in which case thedetectable moiety is an enzyme.

For immunohistochemistry, the tissue sample may be fresh or frozen ormay be embedded in paraffin and fixed with a preservative such asformalin, for example.

7. Cell-Based Assays

Cell-based assays and animal models for immune related diseases can beused to further understand the relationship between the genes andpolypeptides identified herein and the development and pathogenesis ofimmune related disease.

In a different approach, cells of a cell type known to be involved in aparticular immune related disease are transfected with the cDNAsdescribed herein, and the ability of these cDNAs to alter immunefunction is analyzed. Suitable cells can be transfected with the desiredgene, and monitored for immune function activity. Such transfected celllines can then be used to test the ability of poly- or monoclonalantibodies or antibody compositions to alter immune function, forexample to modulate T-cell proliferation or inflammatory cellinfiltration. Cells transfected with the coding sequences of the genesidentified herein can further be used to identify drug candidates forthe treatment of immune related diseases.

In addition, primary cultures derived from transgenic animals (asdescribed below) can be used in the cell-based assays herein, althoughstable cell lines are preferred. Techniques to derive continuous celllines from transgenic animals are well known in the art (see, e.g. Smallet al., Mol. Cell. Biol. 5, 642-648 [1985]).

One suitable cell based assay is the mixed lymphocyte reaction (MLR).Current Protocols in Immunology, unit 3.12; edited by J E Coligan, A MKruisbeek, D H Marglies, E M Shevach, W Strober, National Institutes ofHealth, Published by John Wiley & Sons, Inc. In this assay, the abilityof a test compound to stimulate the proliferation of activated T cellsis assayed. A suspension of responder T cells is cultured with allogenicstimulator cells and the proliferation of T cells is measured by uptakeof tritiated thymidine. This assay is a general measure of T cellreactivity. Since the majority of T cells respond to and produce IL-2upon activation, differences in responsiveness in this assay in partreflect differences in IL-2 production by the responding cells. The MLRresults can be verified by a standard lymphokine (IL-2) detection assay.Current Protocols in Immunology, supra, 3.15, 6.3.

A proliferative T cell response in an MLR assay may be due to amitogenic response or may be due to a stimulatory response by the Tcells. Additional verification of the T cell stimulatory activity of thepolypeptides of the invention can be obtained by a costimulation assay.T cell activation requires an antigen specific signal mediated throughthe major histocompatability complex (MHC) and a costimulatory signalmediated through a second ligand binding interaction, for example, theB7(CD80, CD86)/CD28 binding interaction. CD28 crosslinking increaseslymphokine secretion by activated T cells. T cell activation has bothnegative and positive controls through the binding of ligands which havea negative or positive effect. CD28 and CTLA-4 are related glycoproteinsin the Ig superfamily which bind to B7. CD28 binding to B7 has apositive costimulation effect of T cell activation; conversely, CTLA-4binding to B7 has a negative T cell deactivating effect. Chambers, C. A.and Allison, J. P., Curr. Opin. Immunol. (1997) 9:396. Schwartz, R. H.,Cell (1992) 71:1065; Linsley, P. S, and Ledbetter, J. A., Annu. Rev.Immunol. (1993) 11:191; June, C. H. et al, Immunol. Today (1994) 15:321;Jenkins, M. K., Immunity (1994) 1:443-446.

Polypeptides of the invention, as well as other compounds of theinvention, which are stimulators (costimulators) of T cellproliferation, as determined by MLR assays, for example, are useful intreating immune related diseases characterized by poor, suboptimal orinadequate immune function. These diseases are treated by stimulatingthe proliferation and activation of T cells (and T cell mediatedimmunity) and enhancing the immune response in a mammal throughadministration of a stimulatory compound, such as the stimulatingpolypeptides of the invention. The stimulating polypeptide may be aPRO301, PRO362, PRO245 or PRO1868 polypeptide or an agonist antibodytherefor. Immunoadjuvant therapy for treatment of tumors, described inmore detail below, is an example of this use of the stimulatingcompounds of the invention. Antibodies which bind to inhibitorypolypeptides function to enhance the immune response by removing theinhibitory effect of the inhibiting polypeptides. This effect is seen inexperiments using anti-CTLA-4 antibodies which enhance T cellproliferation, presumably by removal of the inhibitory signal caused byCTLA-4 binding. Walunas, T. L. et al, Immunity (1994) 1:405. This use isalso validated in experiments with 4-1BB glycoprotein, a member of thetumor necrosis factor receptor family which binds to a ligand (4-1BBL)expressed on primed T cells and signals T cell activation and growth.Alderson, M. E. et al., J. Immunol. (1994) 24:2219. Inhibition of 4-1BBbinding by treatment with an anti-4-1BB antibody increases the severityof graft-versus-host disease and may be used to eradicate tumors.Hellstrom, I. and Hellstrom, K. E., Crit. Rev. Immunol. (1998) 18:1.

On the other hand, polypeptides of the invention, such as antagonistantibodies, as well as other compounds of the invention, which areinhibitors of T cell proliferation/activation and/or lymphokinesecretion, can be directly used to suppress the immune response. Thesecompounds are useful to reduce the degree of the immune response and totreat immune related diseases characterized by a hyperactive,superoptimal, or autoimmune response. Alternatively, antibodies whichbind to the stimulating polypeptides of the invention and block thestimulating effect of these molecules can be used to suppress the T cellmediated immune response by inhibiting T cell proliferation/activationand/or lymphokine secretion. Blocking the stimulating effect of thepolypeptides suppresses the immune response of the mammal.

8. Animal Models

The results of the cell based in vitro assays can be further verifiedusing in vivo animal models and assays for T-cell function. A variety ofwell known animal models can be used to further understand the role ofthe genes identified herein in the development and pathogenesis ofimmune related disease, and to test the efficacy of candidatetherapeutic agents, including antibodies, and other antagonists of thenative polypeptides, including small molecule antagonists. The in vivonature of such models makes them particularly predictive of responses inhuman patients. Animal models of immune related diseases include bothnon-recombinant and recombinant (transgenic) animals. Non-recombinantanimal models include, for example, rodent, e.g., murine models. Suchmodels can be generated by introducing cells into syngenic mice usingstandard techniques, e.g. subcutaneous injection, tail vein injection,spleen implantation, intraperitoneal implantation, implantation underthe renal capsule, etc.

Contact hypersensitivity is a simple in vivo assay of cell mediatedimmune function. In this procedure, epidermal cells are exposed toexogenous haptens which give rise to a delayed type hypersensitivityreaction which is measured and quantitated. Contact sensitivity involvesan initial sensitizing phase followed by an elicitation phase. Theelicitation phase occurs when the epidermal cells encounter an antigento which they have had previous contact. Swelling and inflammationoccur, making this an excellent model of human allergic contactdermatitis. A suitable procedure is described in detail in CurrentProtocols in Immunology, Eds. J. E. Cologan, A. M. Kruisbeek, D. H.Margulies, E. M. Shevach and W. Strober, John Wiley & Sons, Inc., 1994,unit 4.2. See also Grabbe, S, and Schwarz, T, Immun. Today 19(1):37-44(1998).

Graft-versus-host disease occurs when immunocompetent cells aretransplanted into immunosuppressed or tolerant patients. The donor cellsrecognize and respond to host antigens. The response can vary from lifethreatening severe inflammation to mild cases of diarrhea and weightloss. Graft-versus-host disease models provide a means of assessing Tcell reactivity against MHC antigens and minor transplant antigens. Asuitable procedure is described in detail in Current Protocols inImmunology, supra, unit 4.3.

An animal model for skin allograft rejection is a means of testing theability of T cells to mediate in vivo tissue destruction which isindicative of and a measure of their role in anti-viral and tumorimmunity. The most common and accepted models use murine tail-skingrafts. Repeated experiments have shown that skin allograft rejection ismediated by T cells, helper T cells and killer-effector T cells, and notantibodies. Auchincloss, H. Jr. and Sachs, D. H., FundamentalImmunology, 2nd ed., W. E. Paul ed., Raven Press, NY, 1989, 889-992. Asuitable procedure is described in detail in Current Protocols inImmunology, supra, unit 4.4. Other transplant rejection models which canbe used to test the compounds of the invention are the allogeneic hearttransplant models described by Tanabe, M. et al, Transplantation (1994)58:23 and Tinubu, S. A. et al, J. Immunol. (1994) 4330-4338.

Animal models for delayed type hypersensitivity provides an assay ofcell mediated immune function as well. Delayed type hypersensitivityreactions are a T cell mediated in vivo immune response characterized byinflammation which does not reach a peak until after a period of timehas elapsed after challenge with an antigen. These reactions also occurin tissue specific autoimmune diseases such as multiple sclerosis (MS)and experimental autoimmune encephalomyelitis (EAE, a model for MS). Asuitable procedure is described in detail in Current Protocols inImmunology, above, unit 4.5.

EAE is a T cell mediated autoimmune disease characterized by T cell andmononuclear cell inflammation and subsequent demyelination of axons inthe central nervous system. EAE is generally considered to be a relevantanimal model for MS in humans. Bolton, C., Multiple Sclerosis (1995)1:143. Both acute and relapsing-remitting models have been developed.The compounds of the invention can be tested for T cell stimulatory orinhibitory activity against immune mediated demyelinating disease usingthe protocol described in Current Protocols in Immunology, above, units15.1 and 15.2. See also the models for myelin disease in whicholigodendrocytes or Schwann cells are grafted into the central nervoussystem as described in Duncan, I. D. et al, Molec. Med. Today (1997)554-561.

An animal model for arthritis is collagen-induced arthritis. This modelshares clinical, histological and immunological characteristics of humanautoimmune rheumatoid arthritis and is an acceptable model for humanautoimmune arthritis. Mouse and rat models are characterized bysynovitis, erosion of cartilage and subchondral bone. The compounds ofthe invention can be tested for activity against autoimmune arthritisusing the protocols described in Current Protocols in Immunology, above,units 15.5. See also the model using a monoclonal antibody to CD18 andVLA-4 integrins described in Issekutz, A. C. et al., Immunology (1996)88:569.

A model of asthma has been described in which antigen-induced airwayhyper-reactivity, pulmonary eosinophilia and inflammation are induced bysensitizing an animal with ovalbumin and then challenging the animalwith the same protein delivered by aerosol. Several animal models(guinea pig, rat, non-human primate) show symptoms similar to atopicasthma in humans upon challenge with aerosol antigens. Murine modelshave many of the features of human asthma. Suitable procedures to testthe compounds of the invention for activity and effectiveness in thetreatment of asthma are described by Wolyniec, W. W. et al, Am. J.Respir. Cell Mol. Biol. (1998) 18:777 and the references cited therein.

Additionally, the compounds of the invention can be tested on animalmodels for psoriasis like diseases. Evidence suggests a T cellpathogenesis for psoriasis. The compounds of the invention can be testedin the scid/scid mouse model described by Schon, M. P. et al, Nat. Med.(1997) 3:183, in which the mice demonstrate histopathologic skin lesionsresembling psoriasis. Another suitable model is the human skin/scidmouse chimera prepared as described by Nickoloff, B. J. et al, Am. J.Path. (1995) 146:580.

Recombinant (transgenic) animal models can be engineered by introducingthe coding portion of the genes identified herein into the genome ofanimals of interest, using standard techniques for producing transgenicanimals. Animals that can serve as a target for transgenic manipulationinclude, without limitation, mice, rats, rabbits, guinea pigs, sheep,goats, pigs, and non-human primates, e.g. baboons, chimpanzees andmonkeys. Techniques known in the art to introduce a transgene into suchanimals include pronucleic microinjection (Hoppe and Wanger, U.S. Pat.No. 4,873,191); retrovirus-mediated gene transfer into germ lines (e.g.,Van der Putten et al., Proc. Natl. Acad. Sci. USA 82, 6148-615 [1985]);gene targeting in embryonic stem cells (Thompson et al., Cell 56,313-321 [1989]); electroporation of embryos (Lo, Mol. Cell. Biol. 3,1803-1814 [1983]); sperm-mediated gene transfer (Lavitrano et al., Cell57, 717-73 [1989]). For review, see, for example, U.S. Pat. No.4,736,866.

For the purpose of the present invention, transgenic animals includethose that carry the transgene only in part of their cells (“mosaicanimals”). The transgene can be integrated either as a single transgene,or in concatamers, e.g., head-to-head or head-to-tail tandems. Selectiveintroduction of a transgene into a particular cell type is also possibleby following, for example, the technique of Lasko et al., Proc. Natl.Acad. Sci. USA 89, 623-636 (1992).

The expression of the transgene in transgenic animals can be monitoredby standard techniques. For example, Southern blot analysis or PCRamplification can be used to verify the integration of the transgene.The level of mRNA expression can then be analyzed using techniques suchas in situ hybridization, Northern blot analysis, PCR, orimmunocytochemistry.

The animals may be further examined for signs of immune diseasepathology, for example by histological examination to determineinfiltration of immune cells into specific tissues. Blocking experimentscan also be performed in which the transgenic animals are treated withthe compounds of the invention to determine the extent of effects on Tcell proliferation. In these experiments, blocking antibodies which bindto the polypeptide of the invention, prepared as described above, areadministered to the animal and the effect on immune function isdetermined.

Alternatively, “knock out” animals can be constructed which have adefective or altered gene encoding a polypeptide identified herein, as aresult of homologous recombination between the endogenous gene encodingthe polypeptide and altered genomic DNA encoding the same polypeptideintroduced into an embryonic cell of the animal. For example, cDNAencoding a particular polypeptide can be used to clone genomic DNAencoding that polypeptide in accordance with established techniques. Aportion of the genomic DNA encoding a particular polypeptide can bedeleted or replaced with another gene, such as a gene encoding aselectable marker which can be used to monitor integration. Typically,several kilobases of unaltered flanking DNA (both at the 5′ and 3′ ends)are included in the vector [see e.g., Thomas and Capecchi, Cell, 51:503(1987) for a description of homologous recombination vectors]. Thevector is introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced DNA has homologouslyrecombined with the endogenous DNA are selected [see e.g., Li et al.,Cell, 69:915 (1992)]. The selected cells are then injected into ablastocyst of an animal (e.g., a mouse or rat) to form aggregationchimeras [see e.g., Bradley, in Teratocarcinomas and Embryonic StemCells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987),pp. 113-152]. A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term tocreate a “knock out” animal. Progeny harboring the homologouslyrecombined DNA in their germ cells can be identified by standardtechniques and used to breed animals in which all cells of the animalcontain the homologously recombined DNA. Knockout animals can becharacterized for instance, for their ability to defend against certainpathological conditions and for their development of pathologicalconditions due to absence of the polypeptide.

9. Immuno Adjuvant Therapy

In one embodiment, compounds of the invention having animmunostimulatory effect can be used in immunoadjuvant therapy for thetreatment of tumors (cancer). It is now well established that T cellsrecognize human tumor specific antigens. One group of tumor antigens,encoded by the MAGE, BAGE and GAGE families of genes, are silent in alladult normal tissues, but are expressed in significant amounts intumors, such as melanomas, lung tumors, head and neck tumors, andbladder carcinomas. DeSmet, C. et al, (1996) Proc. Natl. Acad. Sci. USA,93:7149. It has been shown that costimulation of T cells induces tumorregression and an antitumor response both in vitro and in vivo. Melero,I. et al, Nature Medicine (1997) 3:682; Kwon, E. D. et al, Proc. Natl.Acad. Sci. USA (1997) 94:8099; Lynch, D. H. et al, Nature Medicine(1997) 3:625; Finn, O. J. and Lotze, M. T., J. Immunol. (1998) 21:114.The stimulatory compounds of the invention can be administered asadjuvants, alone or together with a growth regulating agent, cytotoxicagent or chemotherapeutic agent, to stimulate T cellproliferation/activation and an antitumor response to tumor antigens.The growth regulating, cytotoxic, or chemotherapeutic agent may beadministered in conventional amounts using known administration regimes.Immunostimulating activity by the compounds of the invention allowsreduced amounts of the growth regulating, cytotoxic, or chemotherapeuticagents thereby potentially lowering the toxicity to the patient.

Cancer is characterized by the increase in the number of abnormal, orneoplastic, cells derived from a normal tissue which proliferate to forma tumor mass, the invasion of adjacent tissues by these neoplastic tumorcells, and the generation of malignant cells which eventually spread viathe blood or lymphatic system to regional lymph nodes and to distantsites (metastasis). In a cancerous state a cell proliferates underconditions in which normal cells would not grow. Cancer manifests itselfin a wide variety of forms, characterized by different degrees ofinvasiveness and aggressiveness.

Alteration of gene expression is intimately related to the uncontrolledcell growth and de-differentiation which are a common feature of allcancers. The genomes of certain well studied tumors have been found toshow decreased expression of recessive genes, usually referred to astumor suppression genes, which would normally function to preventmalignant cell growth, and/or overexpression of certain dominant genes,such as oncogenes, that act to promote malignant growth. Each of thesegenetic changes appears to be responsible for importing some of thetraits that, in aggregate, represent the full neoplastic phenotype(Hunter, Cell 64, 1129 [1991]; Bishop, Cell 64, 235-248 [1991]).

A well known mechanism of gene (e.g. oncogene) overexpression in cancercells is gene amplification. This is a process where in the chromosomeof the ancestral cell multiple copies of a particular gene are produced.The process involves unscheduled replication of the region of chromosomecomprising the gene, followed by recombination of the replicatedsegments back into the chromosome (Alitalo et al., Adv. Cancer Res. 47,235-281 [1986]). It is believed that the overexpression of the geneparallels gene amplification, i.e. is proportionate to the number ofcopies made.

Proto-oncogenes that encode growth factors and growth factor receptorshave been identified to play important roles in the pathogenesis ofvarious human malignancies, including breast cancer. For example, it hasbeen found that the human ErbB2 gene (erbB2, also known as her2, orc-erbB-2), which encodes a 185-kd transmembrane glycoprotein receptor(p185^(HER2); HER2) related to the epidermal growth factor receptor(EGFR), is overexpressed in about 25% to 30% of human breast cancer(Slamon et al., Science 235:177-182 [1987]; Slamon et al., Science244:707-712 [1989]).

It has been reported that gene amplification of a protooncogene is anevent typically involved in the more malignant forms of cancer, andcould act as a predictor of clinical outcome (Schwab et al., GenesChromosomes Cancer 1, 181-193 [1990]; Alitalo et al., supra). Thus,erbB2 overexpression is commonly regarded as a predictor of a poorprognosis, especially in patients with primary disease that involvesaxillary lymph nodes (Slamon et al., [1987] and [1989], supra; Ravdinand Chamness, Gene 159:19-27 [1995]; and Hynes and Stem, Biochim BiophysActa 1198:165-184 [1994]), and has been linked to sensitivity and/orresistance to hormone therapy and chemotherapeutic regimens, includingCMF (cyclophosphamide, methotrexate, and fluoruracil) and anthracyclines(Baselga et al., Oncology 11 (3 Suppl 1):43-48 [1997]). However, despitethe association of erbB2 overexpression with poor prognosis, the odds ofHER2-positive patients responding clinically to treatment with taxaneswere greater than three times those of HER2-negative patients (Ibid). Arecombinant humanized anti-ErbB2 (anti-HER2) monoclonal antibody (ahumanized version of the murine anti-ErbB2 antibody 4D5, referred to asrhuMAb HER2 or Herceptin7) has been clinically active in patients withErbB2-overexpressing metastatic breast cancers that had receivedextensive prior anticancer therapy. (Baselga et al., J. Clin. Oncol.14:737-744 [1996]).

10. Screening Assays for Drug Candidates

Screening assays for drug candidates are designed to identify compoundsthat bind or complex with the polypeptides encoded by the genesidentified herein or a biologically active fragment thereof, orinterfere with the expression and/or activity of the polypeptidesencoded by genes identified herein or with the interaction of theencoded polypeptides with other cellular proteins. Such screening assayswill include assays amenable to high-throughput screening of chemicallibraries, making them particularly suitable for identifying smallmolecule drug candidates. Small molecules contemplated include syntheticorganic or inorganic compounds, including peptides, preferably solublepeptides, (poly)peptide-immunoglobulin fusions, and, in particular,antibodies including, without limitation, poly- and monoclonalantibodies and antibody fragments, single-chain antibodies,anti-idiotypic antibodies, and chimeric or humanized versions of suchantibodies or fragments, as well as human antibodies and antibodyfragments. The assays can be performed in a variety of formats,including protein-protein binding assays, biochemical screening assays,immunoassays and cell based assays, which are well characterized in theart.

All assays are common in that they call for contacting the drugcandidate with a polypeptide encoded by a nucleic acid identified hereinunder conditions and for a time sufficient to allow these two componentsto interact.

In binding assays, the interaction is binding and the complex formed canbe isolated or detected in the reaction mixture. In a particularembodiment, the polypeptide encoded by the gene identified herein or thedrug candidate is immobilized on a solid phase, e.g. on a microtiterplate, by covalent or non-covalent attachments. Non-covalent attachmentgenerally is accomplished by coating the solid surface with a solutionof the polypeptide and drying. Alternatively, an immobilized antibody,e.g. a monoclonal antibody, specific for the polypeptide to beimmobilized can be used to anchor it to a solid surface. The assay isperformed by adding the non-immobilized component, which may be labeledby a detectable label, to the immobilized component, e.g. the coatedsurface containing the anchored component. When the reaction iscomplete, the non-reacted components are removed, e.g. by washing, andcomplexes anchored on the solid surface are detected. When theoriginally non-immobilized component carries a detectable label, thedetection of label immobilized on the surface indicates that complexingoccurred. Where the originally non-immobilized component does not carrya label, complexing can be detected, for example, by using a labeledantibody specifically binding the immobilized complex.

If the candidate compound interacts with but does not bind to aparticular protein encoded by a gene identified herein, its interactionwith that protein can be assayed by methods well known for detectingprotein-protein interactions. Such assays include traditionalapproaches, such as, cross-linking, co-immunoprecipitation, andco-purification through gradients or chromatographic columns. Inaddition, protein-protein interactions can be monitored by using ayeast-based genetic system described by Fields and co-workers [Fieldsand Song, Nature (London) 340, 245-246 (1989); Chien et al., Proc. Natl.Acad. Sci. USA 88, 9578-9582 (1991)] as disclosed by Chevray and Nathans[Proc. Natl. Acad. Sci. USA 89, 5789-5793 (1991)]. Many transcriptionalactivators, such as yeast GAL4, consist of two physically discretemodular domains, one acting as the DNA-binding domain, while the otherone functioning as the transcription activation domain. The yeastexpression system described in the foregoing publications (generallyreferred to as the “two-hybrid system”) takes advantage of thisproperty, and employs two hybrid proteins, one in which the targetprotein is fused to the DNA-binding domain of GAL4, and another, inwhich candidate activating proteins are fused to the activation domain.The expression of a GAL1-lacZ reporter gene under control of aGAL4-activated promoter depends on reconstitution of GAL4 activity viaprotein-protein interaction. Colonies containing interactingpolypeptides are detected with a chromogenic substrate forβ-galactosidase. A complete kit (MATCHMAKER™) for identifyingprotein-protein interactions between two specific proteins using thetwo-hybrid technique is commercially available from Clontech. Thissystem can also be extended to map protein domains involved in specificprotein interactions as well as to pinpoint amino acid residues that arecrucial for these interactions.

In order to find compounds that interfere with the interaction of a geneidentified herein and other intra- or extracellular components can betested, a reaction mixture is usually prepared containing the product ofthe gene and the intra- or extracellular component under conditions andfor a time allowing for the interaction and binding of the two products.To test the ability of a test compound to inhibit binding, the reactionis run in the absence and in the presence of the test compound. Inaddition, a placebo may be added to a third reaction mixture, to serveas positive control. The binding (complex formation) between the testcompound and the intra- or extracellular component present in themixture is monitored as described above. The formation of a complex inthe control reaction(s) but not in the reaction mixture containing thetest compound indicates that the test compound interferes with theinteraction of the test compound and its reaction partner.

11. Compositions and Methods for the Treatment of Immune RelatedDiseases

The compositions useful in the treatment of immune related diseasesinclude, without limitation, antibodies, small organic and inorganicmolecules, peptides, phosphopeptides, antisense and ribozyme molecules,triple helix molecules, etc. that inhibit or stimulate immune function,for example, T cell proliferation/activation, lymphokine release, orimmune cell infiltration, depends on the disease to be treated.

For example, antisense RNA and RNA molecule act to directly block thetranslation of mRNA by hybridizing to targeted mRNA and preventingprotein translation. When antisense DNA is used,oligodeoxyribonucleotides derived from the translation initiation site,e.g. between about −10 and +10 positions of the target gene nucleotidesequence, are preferred.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. Ribozymes act by sequence-specific hybridization to thecomplementary target RNA, followed by endonucleolytic cleavage. Specificribozyme cleavage sites within a potential RNA target can be identifiedby known techniques. For further details see, e.g. Rossi, CurrentBiology 4, 469-471 (1994), and PCT publication No. WO 97/33551(published Sep. 18, 1997).

Nucleic acid molecules in triple helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple helix formation via Hoogsteen basepairing rules, which generally require sizeable stretches of purines orpyrimidines on one strand of a duplex. For further details see, e.g. PCTpublication No. WO 97/33551, supra.

These molecules can be identified by any or any combination of thescreening assays discussed above and/or by any other screeningtechniques well known for those skilled in the art.

12. Antibodies

Among the most promising drug candidates according to the presentinvention are antibodies and antibody fragments which may inhibit(antagonists) or stimulate (agonists) T cell proliferation, leucocyteinfiltration, etc. Exemplary antibodies include polyclonal, monoclonal,humanized, bispecific and heteroconjugate antibodies.

a. Polyclonal Antibodies

Methods of preparing polyclonal antibodies are known to skilled artisan.Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent, and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Theimmunizing agent may include the PRO301, PRO362, PRO245 or PRO1868polypeptide of the invention or a fragment or fusion protein thereof. Itmay be useful to conjugate the immunizing agent to a protein known to beimmunogenic in the mammal being immunized. Examples of such immunogenicproteins include but are not limited to keyhole limpet hemocyanin, serumalbumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examplesof adjuvants which may be employed include Freund's complete adjuvantand MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalosedicorynomycolate). The immunization protocol may be selected by oneskilled in the art without undue experimentation.

b. Monoclonal Antibodies

Antibodies which recognize and bind to the polypeptides of the inventionor which act as antagonists thereto may, alternatively be monoclonalantibodies. Monoclonal antibodies may be prepared using hybridomamethods, such as those described by Kohler and Milstein, Nature, 256:495(1975). In a hybridoma method, a mouse, hamster, or other appropriatehost animal, is typically immunized with an immunizing agent to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The immunizing agent will typically include the PRO301, PRO362, PRO245or PRO1868 polypeptide of the invention, an antigenic fragment or afusion protein thereof. Generally, either peripheral blood lymphocytes(“PBLs”) are used if cells of human origin are desired, or spleen cellsor lymph node cells are used if non-human mammalian sources are desired.The lymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell [Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59-103]. Immortalized cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine and human origin. Usually, rat or mouse myeloma cell lines areemployed. The hybridoma cells may be cultured in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, immortalized cells. For example, ifthe parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (“HATmedium”), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Rockville, Md. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against thepolypeptide of the invention or having similar activity as thepolypeptide of the invention. Preferably, the binding specificity ofmonoclonal antibodies produced by the hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchardanalysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods[Goding, supra]. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxyapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences [U.S. Pat.No. 4,816,567; Morrison et al., supra] or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies are preferably monovalent antibodies. Methods forpreparing monovalent antibodies are well known in the art. For example,one method involves recombinant expression of immunoglobulin light chainand modified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart.

c. Human and Humanized Antibodies

The antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and coworkers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985); Boerner et al., J. Immunol., 147(1):86-95 (1991); U.S. Pat. No.5,750,373]. Similarly, human antibodies can be made by introducing ofhuman immunoglobulin loci into transgenic animals, e.g., mice in whichthe endogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779-783(1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368,812-13 (1994); Fishwild et al., Nature _(—) Biotechnology 14, 845-51(1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg andHuszar, Intern. Rev. Immunol. 13 65-93 (1995).

d. Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities may befor the polypeptide of the invention, the other one is for any otherantigen, and preferably for a cell-surface protein or receptor orreceptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the coexpression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305:537-539 [1983]). Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are cotransfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

e. Heteroconjugate Antibodies

Heteroconjugate antibodies are composed of two covalently joinedantibodies. Such antibodies have, for example, been proposed to targetimmune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and fortreatment of HIV infection (WO 91/00360; WO 92/200373; EP 03089). It iscontemplated that the antibodies may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S.Pat. No. 4,676,980.

f. Effect or Function Engineering

It may be desirable to modify the antibody of the invention with respectto effector function, so as to enhance the effectiveness of the antibodyin treating an immune related disease, for example. For example cysteineresidue(s) may be introduced in the Fc region, thereby allowinginterchain disulfide bond formation in this region. The homodimericantibody thus generated may have improved internalization capabilityand/or increased complement-mediated cell killing and antibody-dependentcellular cytotoxicity (ADCC). See Caron et al., J. Exp Med.176:1191-1195 (1992) and Shopes, B., J. Immunol. 148:2918-2922 (1992).Homodimeric antibodies with enhanced anti-tumor activity may also beprepared using heterobifunctional cross-linkers as described in Wolff etal. Cancer Research 53:2560-2565 (1993). Alternatively, an antibody canbe engineered which has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,Anti-Cancer Drug Design, 3:219-230 (1989).

g. Immunoconjugates

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin(e.g. an enzymatically active toxin of bacterial, fungal, plant oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof which can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y and¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such asbis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

In another embodiment, the antibody may be conjugated to a “receptor”(such streptavidin) for utilization in tissue pretargeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g. avidin) which is conjugatedto a cytotoxic agent (e.g. a radionucleotide).

h. Immunoliposomes

The proteins, antibodies, etc. disclosed herein may also be formulatedas immunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl. Acad.Sci. USA, 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem. 257: 286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent (such as doxorubicin) may be optionally containedwithin the liposome. See Gabizon et al., J. National Cancer Inst. 81(19) 1484 (1989).

13. Pharmaceutical Compositions

The active molecules of the invention, including polypeptides andantibodies, as well as other molecules identified by the screeningassays disclosed above, can be administered for the treatment ofinflammatory diseases, in the form of pharmaceutical compositions.

Therapeutic formulations of the active molecule, preferably a PRO301,PRO362, PRO245 or PRO1868 polypeptide or antibody of the invention, areprepared for storage by mixing the active molecule having the desireddegree of purity with optional pharmaceutically acceptable carriers,excipients or stabilizers (Remington's Pharmaceutical Sciences 16thedition, Osol, A. Ed. [1980]), in the form of lyophilized formulationsor aqueous solutions. Acceptable carriers, excipients, or stabilizersare nontoxic to recipients at the dosages and concentrations employed,and include buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Compounds identified by the screening assays of the present inventioncan be formulated in an analogous manner, using standard techniques wellknown in the art.

Lipofections or liposomes can also be used to deliver the polypeptide,antibody, or an antibody fragment, into cells. Where antibody fragmentsare used, the smallest fragment which specifically binds to the bindingdomain of the target protein is preferred. For example, based upon thevariable region sequences of an antibody, peptide molecules can bedesigned which retain the ability to bind the target protein sequence.Such peptides can be synthesized chemically and/or produced byrecombinant DNA technology (see, e.g. Marasco et al., Proc. Natl. Acad.Sci. USA 90, 7889-7893 [1993]).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Alternatively, or in addition, the composition may comprise a cytotoxicagent, cytokine or growth inhibitory agent. Such molecules are suitablypresent in combination in amounts that are effective for the purposeintended.

The active molecules may also be entrapped in microcapsules prepared,for example, by coascervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37 C, resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

14. Methods of Treatment

It is contemplated that the polypeptides, antibodies and other activecompounds of the present invention may be used to treat variousinflammatory diseases and conditions, such as T cell mediated diseases,including those characterized by infiltration of leucocyte cells into atissue, stimulation of T-cell proliferation, inhibition of T-cellproliferation, increased or decreased vascular permeability or theinhibition thereof.

PRO301, PRO362, PRO245 and PRO1868 encode new members of a family ofproteins characterized by homology to A33 antigen. The proinflammatorynature of these polypeptides is indicated in the in vitro assaysdescribed below. Accordingly, antagonists of these polypeptides would beuseful to treat inflammatory diseases.

PRO301, PRO362, PRO245 and PRO1868 (SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 9 and SEQ ID NO: 31, respectively), share homology with junctionaladhesion molecule (JAM), Martin-Padura et al., J. Cell Biol. 1998142(1): 117-27. The most substantial identity is shared by the PRO301protein encoded by DNA40628 (SEQ ID NO: 1) at 67%. JAM is involved inthe recruitment of monocytes in response to MCP-1, MCP-3 and LPS invivo. Antibodies to JAM block monocyte transmigration in vivo. JAM islocalized to the murine epithelia and endothelia as a junctionaladhesion molecule for monocyte transmigration. Other leukocytes may alsouse JAM, but no information supports this notion. JAM is elevated in thecolon of mice with colitis and likely plays a role in the recruitment ofmonocytes or leukocytes into the colonic lesion.

Exemplary conditions or disorders to be treated with antagonists ofPRO301, PRO362, PRO245 or PRO1868 polypeptides, antibodies and othercompounds of the invention, include, but are not limited to,inflammatory bowel disease (i.e., ulcerative colitis, Crohn's disease),systemic lupus erythematosus, rheumatoid arthritis, juvenile chronicarthritis, spondyloarthropathies, systemic sclerosis (scleroderma),idiopathic inflammatory myopathies (dermatomyositis, polymyositis),Sjögren's syndrome, systemic vasculitis, sarcoidosis, autoimmunehemolytic anemia (immune pancytopenia, paroxysmal nocturnalhemoglobinuria), autoimmune thrombocytopenia (idiopathicthrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis(Grave's disease, Hashimoto's thyroiditis, juvenile lymphocyticthyroiditis, atrophic thyroiditis), diabetes mellitus, immune-mediatedrenal disease (glomerulonephritis, tubulointerstitial nephritis),demyelinating diseases of the central and peripheral nervous systemssuch as multiple sclerosis, idiopathic demyelinating polyneuropathy orGuillain-Barré syndrome, and chronic inflammatory demyelinatingpolyneuropathy, hepatobiliary diseases such as infectious hepatitis(hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmunechronic active hepatitis, primary biliary cirrhosis, granulomatoushepatitis, and sclerosing cholangitis, inflammatory and fibrotic lungdiseases such as cystic fibrosis, gluten-sensitive enteropathy, andWhipple's disease, autoimmune or immune-mediated skin diseases includingbullous skin diseases, erythema multiforme and contact dermatitis,psoriasis, allergic diseases such as asthma, allergic rhinitis, atopicdermatitis, food hypersensitivity and urticaria, immunologic diseases ofthe lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosisand hypersensitivity pneumonitis, transplantation associated diseasesincluding graft rejection and graft-versus-host-disease.

In systemic lupus erythematosus, the central mediator of disease is theproduction of auto-reactive antibodies to self proteins/tissues and thesubsequent generation of immune-mediated inflammation. Antibodies eitherdirectly or indirectly mediate tissue injury. Though T lymphocytes havenot been shown to be directly involved in tissue damage, T lymphocytesare required for the development of auto-reactive antibodies. Thegenesis of the disease is thus T lymphocyte dependent. Multiple organsand systems are affected clinically including kidney, lung,musculoskeletal system, mucocutaneous, eye, central nervous system,cardiovascular system, gastrointestinal tract, bone marrow and blood.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune inflammatorydisease that mainly involves the synovial membrane of multiple jointswith resultant injury to the articular cartilage. The pathogenesis is Tlymphocyte dependent and is associated with the production of rheumatoidfactors, auto-antibodies directed against self IgG, with the resultantformation of immune complexes that attain high levels in joint fluid andblood. These complexes in the joint may induce the marked infiltrate oflymphocytes and monocytes into the synovium and subsequent markedsynovial changes; the joint space/fluid is infiltrated by similar cellswith the addition of numerous neutrophils. Tissues affected areprimarily the joints, often in symmetrical pattern. However,extra-articular disease also occurs in two major forms. One form is thedevelopment of extra-articular lesions with ongoing progressive jointdisease and typical lesions of pulmonary fibrosis, vasculitis, andcutaneous ulcers. The second form of extra-articular disease is the socalled Felty's syndrome which occurs late in the RA disease course,sometimes after joint disease has become quiescent, and involves thepresence of neutropenia, thrombocytopenia and splenomegaly. This can beaccompanied by vasculitis in multiple organs with formations ofinfarcts, skin ulcers and gangrene. Patients often also developrheumatoid nodules in the subcutis tissue overlying affected joints; thenodules late stages have necrotic centers surrounded by a mixedinflammatory cell infiltrate. Other manifestations which can occur in RAinclude: pericarditis, pleuritis, coronary arteritis, interstitialpneumonitis with pulmonary fibrosis, keratoconjunctivitis sicca, andrheumatoid nodules.

Juvenile chronic arthritis is a chronic idiopathic inflammatory diseasewhich begins often at less than 16 years of age. Its phenotype has somesimilarities to RA; some patients which are rheumatoid factor positiveare classified as juvenile rheumatoid arthritis. The disease issub-classified into three major categories: pauciarticular,polyarticular, and systemic. The arthritis can be severe and istypically destructive and leads to joint ankylosis and retarded growth.Other manifestations can include chronic anterior uveitis and systemicamyloidosis.

Spondyloarthropathies are a group of disorders with some common clinicalfeatures and the common association with the expression of HLA-B27 geneproduct. The disorders include: ankylosing spondylitis, Reiter'ssyndrome (reactive arthritis), arthritis associated with inflammatorybowel disease, spondylitis associated with psoriasis, juvenile onsetspondyloarthropathy and undifferentiated spondyloarthropathy.Distinguishing features include sacroileitis with or withoutspondylitis; inflammatory asymmetric arthritis; association with HLA-B27(a serologically defined allele of the HLA-B locus of class I MHC);ocular inflammation, and absence of autoantibodies associated with otherrheumatoid disease. The cell most implicated as key to induction of thedisease is the CD8+ T lymphocyte, a cell which targets antigen presentedby class I MHC molecules. CD8+ T cells may react against the class I MHCallele HLA-B27 as if it were a foreign peptide expressed by MHC class 1molecules. It has been hypothesized that an epitope of HLA-B27 may mimica bacterial or other microbial antigenic epitope and thus induce a CD8+T cells response.

Systemic sclerosis (scleroderma) has an unknown etiology. A hallmark ofthe disease is induration of the skin; likely this is induced by anactive inflammatory process. Scleroderma can be localized or systemic;vascular lesions are common and endothelial cell injury in themicrovasculature is an early and important event in the development ofsystemic sclerosis; the vascular injury may be immune mediated. Animmunologic basis is implied by the presence of mononuclear cellinfiltrates in the cutaneous lesions and the presence of anti-nuclearantibodies in many patients. ICAM-1 is often upregulated on the cellsurface of fibroblasts in skin lesions suggesting that T cellinteraction with these cells may have a role in the pathogenesis of thedisease. Other organs involved include: the gastrointestinal tract:smooth muscle atrophy and fibrosis resulting in abnormalperistalsis/motility; kidney: concentric subendothelial intimalproliferation affecting small arcuate and interlobular arteries withresultant reduced renal cortical blood flow, results in proteinuria,azotemia and hypertension; skeletal muscle: atrophy, interstitialfibrosis; inflammation; lung: interstitial pneumonitis and interstitialfibrosis; and heart: contraction band necrosis, scarring/fibrosis.

Idiopathic inflammatory myopathies including dermatomyositis,polymyositis and others are disorders of chronic muscle inflammation ofunknown etiology resulting in muscle weakness. Muscleinjury/inflammation is often symmetric and progressive. Autoantibodiesare associated with most forms. These myositis-specific autoantibodiesare directed against and inhibit the function of components, proteinsand RNA's, involved in protein synthesis.

Sjögren's syndrome is due to immune-mediated inflammation and subsequentfunctional destruction of the tear glands and salivary glands. Thedisease can be associated with or accompanied by inflammatory connectivetissue diseases. The disease is associated with autoantibody productionagainst Ro and La antigens, both of which are small RNA-proteincomplexes. Lesions result in keratoconjunctivitis sicca, xerostomia,with other manifestations or associations including bilary cirrhosis,peripheral or sensory neuropathy, and palpable purpura.

Systemic vasculitis includes diseases in which the primary lesion isinflammation and subsequent damage to blood vessels which results inischemia/necrosis/degeneration to tissues supplied by the affectedvessels and eventual end-organ dysfunction in some cases. Vasculitidescan also occur as a secondary lesion or sequelae to otherimmune-inflammatory mediated diseases such as rheumatoid arthritis,systemic sclerosis, etc., particularly in diseases also associated withthe formation of immune complexes. Diseases in the primary systemicvasculitis group include: systemic necrotizing vasculitis: polyarteritisnodosa, allergic angiitis and granulomatosis, polyangiitis; Wegener'sgranulomatosis; lymphomatoid granulomatosis; and giant cell arteritis.Miscellaneous vasculitides include: mucocutaneous lymph node syndrome(MLNS or Kawasaki's disease), isolated CNS vasculitis, Behet's disease,thromboangiitis obliterans (Buerger's disease) and cutaneous necrotizingvenulitis. The pathogenic mechanism of most of the types of vasculitislisted is believed to be primarily due to the deposition ofimmunoglobulin complexes in the vessel wall and subsequent induction ofan inflammatory response either via ADCC, complement activation, orboth.

Sarcoidosis is a condition of unknown etiology which is characterized bythe presence of epithelioid granulomas in nearly any tissue in the body;involvement of the lung is most common. The pathogenesis involves thepersistence of activated macrophages and lymphoid cells at sites of thedisease with subsequent chronic sequelae resultant from the release oflocally and systemically active products released by these cell types.

Autoimmune hemolytic anemia including autoimmune hemolytic anemia,immune pancytopenia, and paroxysmal noctural hemoglobinuria is a resultof production of antibodies that react with antigens expressed on thesurface of red blood cells (and in some cases other blood cellsincluding platelets as well) and is a reflection of the removal of thoseantibody coated cells via complement mediated lysis and/orADCC/Fc-receptor-mediated mechanisms.

In autoimmune thrombocytopenia including thrombocytopenic purpura, andimmune-mediated thrombocytopenia in other clinical settings, plateletdestruction/removal occurs as a result of either antibody or complementattaching to platelets and subsequent removal by complement lysis, ADCCor FC-receptor mediated mechanisms.

Thyroiditis including Grave's disease, Hashimoto's thyroiditis, juvenilelymphocytic thyroiditis, and atrophic thyroiditis, are the result of anautoimmune response against thyroid antigens with production ofantibodies that react with proteins present in and often specific forthe thyroid gland. Experimental models exist including spontaneousmodels: rats (BUF and BB rats) and chickens (obese chicken strain);inducible models: immunization of animals with either thyroglobulin,thyroid microsomal antigen (thyroid peroxidase).

Type I diabetes mellitus or insulin-dependent diabetes is the autoimmunedestruction of pancreatic islet β cells; this destruction is mediated byauto-antibodies and auto-reactive T cells. Antibodies to insulin or theinsulin receptor can also produce the phenotype ofinsulin-non-responsiveness.

Immune mediated renal diseases, including glomerulonephritis andtubulointerstitial nephritis, are the result of antibody or T lymphocytemediated injury to renal tissue either directly as a result of theproduction of autoreactive antibodies or T cells against renal antigensor indirectly as a result of the deposition of antibodies and/or immunecomplexes in the kidney that are reactive against other, non-renalantigens. Thus other immune-mediated diseases that result in theformation of immune-complexes can also induce immune mediated renaldisease as an indirect sequelae. Both direct and indirect immunemechanisms result in inflammatory response that produces/induces lesiondevelopment in renal tissues with resultant organ function impairmentand in some cases progression to renal failure. Both humoral andcellular immune mechanisms can be involved in the pathogenesis oflesions.

Demyelinating diseases of the central and peripheral nervous systems,including Multiple Sclerosis; idiopathic demyelinating polyneuropathy orGuillain-Barr syndrome; and Chronic Inflammatory DemyelinatingPolyneuropathy, are believed to have an autoimmune basis and result innerve demyelination as a result of damage caused to oligodendrocytes orto myelin directly. In MS there is evidence to suggest that diseaseinduction and progression is dependent on T lymphocytes. MultipleSclerosis is a demyelinating disease that is T lymphocyte-dependent andhas either a relapsing-remitting course or a chronic progressive course.The etiology is unknown; however, viral infections, geneticpredisposition, environment, and autoimmunity all contribute. Lesionscontain infiltrates of predominantly T lymphocyte mediated, microglialcells and infiltrating macrophages; CD4+ T lymphocytes are thepredominant cell type at lesions. The mechanism of oligodendrocyte celldeath and subsequent demyelination is not known but is likely Tlymphocyte driven.

Inflammatory and Fibrotic Lung Disease, including eosinophilicpneumonia, idiopathic pulmonary fibrosis and hypersensitivitypneumonitis may involve a disregulated immune-inflammatory response.Inhibition of that response would be of therapeutic benefit.

Autoimmune or Immune-mediated Skin Disease including Bullous SkinDiseases, Erythema Multiforme, and Contact Dermatitis are mediated byauto-antibodies, the genesis of which is T lymphocyte-dependent.

Psoriasis is a T lymphocyte-mediated inflammatory disease. Lesionscontain infiltrates of T lymphocytes, macrophages and antigen processingcells, and some neutrophils.

Allergic diseases, including asthma; allergic rhinitis; atopicdermatitis; food hypersensitivity; and urticaria are T lymphocytedependent. These diseases are predominantly mediated by T lymphocyteinduced inflammation, IgE mediated-inflammation or a combination ofboth.

Transplantation associated diseases, including Graft rejection andGraft-Versus-Host-Disease (GVHD) are T lymphocyte-dependent; inhibitionof T lymphocyte function is ameliorative.

Patients suffering from other diseases may benefit from enhancement ofthe immune and/or inflammatory response. Such diseases include, but arenot limited to viral infection (including but not limited to AIDS,hepatitis A, B, C, D, E) bacterial infection, fungal infections, andprotozoan and parasitic infections (molecules or derivatives/agonistswhich stimulate the MLR can be utilized therapeutically to enhance theimmune response to infectious agents), diseases of immunodeficiency,including inherited, acquired, infectious induced (as in HIV infection),or iatrogenic (i.e. as from chemotherapy) immunodeficiency, andneoplasia.

It has been demonstrated that some human cancer patients develop anantibody and/or T lymphocyte response to antigens on neoplastic cells.It has also been shown in animal models of neoplasia that enhancement ofthe immune response can result in rejection or regression of thatparticular neoplasm. Molecules that affect the T lymphocyte response inthe MLR have utility in vivo in altering the immune response againstneoplasia.

The inhibition of molecules with proinflammatory properties may alsohave therapeutic benefit in reperfusion injury; stroke; myocardialinfarction; atherosclerosis; acute lung injury; hemorrhagic shock; burn;sepsis/septic shock; acute tubular necrosis; endometriosis; degenerativejoint disease and pancreatitis.

PRO301, PRO362 and PRO245 polypeptides are active as stimulators of theproliferation of stimulated T-lymphocytes (Example 5). Thus, antagonistsof PRO301, PRO362 and PRO245 would be useful in treating immune relateddisorders, particularly inflammatory disorders, such as by inhibitingthe stimulatory effect of PRO301, PRO362 and PRO245 polypeptides. On theother hand, the PRO301, PRO362 and PRO245 polypeptides and agoniststhereof would be useful in treating disorders that benefit fromstimulation of an inflammatory response.

PRO1868 polypeptides of the invention induced redifferentiation ofchondrocytes (Example 19). Thus, PRO1868 and agonists of PRO1868 may beused in the treatment of various bone and/or cartilage relateddisorders.

The PRO301, PRO362, PRO245 and PRO1868 polypeptides, antibodies andother compounds of the present invention are administered to a mammal,preferably a human, in accord with known methods, such as intravenousadministration as a bolus or by continuous infusion over a period oftime, by intramuscular, intraperitoneal, intracerebrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation (intranasal, intrapulmonary) routes. Intravenousor inhaled administration of polypeptides and antibodies is preferred.

In immunoadjuvant therapy, other therapeutic regimens, suchadministration of an anti-cancer agent, may be combined with theadministration of the proteins, antibodies or compounds of the instantinvention. For example, the patient to be treated with theimmunoadjuvants of the invention may also receive an anti-cancer agent(chemotherapeutic agent) or radiation therapy. Preparation and dosingschedules for such chemotherapeutic agents may be used according tomanufacturers' instructions or as determined empirically by the skilledpractitioner. Preparation and dosing schedules for such chemotherapy arealso described in Chemotherapy Service Ed., M. C. Perry, Williams &Wilkins, Baltimore, Md. (1992). The chemotherapeutic agent may precede,or follow administration of the immunoadjuvant or may be givensimultaneously therewith. Additionally, an anti-oestrogen compound suchas tamoxifen or an anti-progesterone such as onapristone (see, EP616812) may be given in dosages known for such molecules.

It may be desirable to also administer antibodies against other immunedisease associated or tumor associated antigens, such as, withoutlimitation, antibodies which bind to CD20, CD11a, CD18, ErbB2, EGFR,ErbB3, ErbB4, or vascular endothelial factor (VEGF). Alternatively, orin addition, two or more antibodies binding the same or two or moredifferent antigens disclosed herein may be coadministered to thepatient. Sometimes, it may be beneficial to also administer one or morecytokines to the patient. In one embodiment, the polypeptides or othercompounds of the invention are coadministered with a growth inhibitoryagent. For example, the growth inhibitory agent may be administeredfirst, followed by a polypeptide or other compound of the invention.However, simultaneous administration or administration first is alsocontemplated. Suitable dosages for the growth inhibitory agent are thosepresently used and may be lowered due to the combined action (synergy)of the growth inhibitory agent and the polypeptide or other compound ofthe invention.

For the treatment or reduction in the severity of immune relateddisease, the appropriate dosage of an a compound of the invention willdepend on the type of disease to be treated, as defined above, theseverity and course of the disease, whether the agent is administeredfor preventive or therapeutic purposes, previous therapy, the patient'sclinical history and response to the compound, and the discretion of theattending physician. The compound is suitably administered to thepatient at one time or over a series of treatments. Preferably, it isdesirable to determine the dose-response curve and the pharmaceuticalcomposition of the invention first in vitro, and then in useful animalmodels prior to testing in humans.

For example, depending on the type and severity of the disease, about 1μg/kg to 15 mg/kg (e.g. 0.1-20 mg/kg) of polypeptide or antibody is aninitial candidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. A typical daily dosage might range from about 1 μg/kg to 100mg/kg or more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment is sustained until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful. Theprogress of this therapy is easily monitored by conventional techniquesand assays.

15. Articles of Manufacture

In another embodiment of the invention, an article of manufacturecontaining materials useful for the diagnosis or treatment of thedisorders described above is provided. The article of manufacturecomprises a container and a label. Suitable containers include, forexample, bottles, vials, syringes, and test tubes. The containers may beformed from a variety of materials such as glass or plastic. Thecontainer holds a composition, which is effective for diagnosing ortreating the condition, and may have a sterile access port (for examplethe container may be an intravenous solution bag or a vial having astopper pierceable by a hypodermic injection needle). The active agentin the composition is usually a polypeptide or an antibody of theinvention. The label on, or associated with, the container indicatesthat the composition is used for diagnosing or treating the condition ofchoice, particularly an immune related condition. The article ofmanufacture may further comprise a second container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use.

16. Diagnosis and Prognosis of Disease

Cell surface proteins, such as proteins, which are overexpressed incertain immune related diseases, are excellent targets for drugcandidates or disease treatment. The same proteins along with secretedproteins encoded by the genes amplified in immune related disease statesfind additional use in the diagnosis and prognosis of these diseases.For example, antibodies directed against the protein products of genesamplified in multiple sclerosis, rheumatoid arthritis, or another immunerelated disease, can be used as diagnostics or prognostics. Suchantibodies and a carrier (e.g., a buffer) may be included in adiagnostic kit in suitable packaging along with instructions for usingthe antibody to detect the protein product.

PRO1868 polypeptides were significantly overexpressed in various humantumor tissues (Example 20), for example lung and breast tumors. Thus,PRO1868 antibodies may be used to diagnose tumors in patients.

The expression of PRO362 polypeptides was found to be significantlyincreased in tissues associated with neoplasia, as well as inflammatorydisease. The expression of PRO245 polypeptides was also significantlyincreased in tissues with chronic inflammatory diseases and neoplasms.Thus, PRO362 and PRO235 antibodies to diagnose inflammed tissues andneoplasms.

For example, antibodies, including antibody fragments, can be used toqualitatively or quantitatively detect the expression of proteinsencoded by the overexpressed or highly expressed genes. The antibodypreferably is equipped with a detectable, e.g. fluorescent label, andbinding can be monitored by light microscopy, flow cytometry,fluorimetry, or other techniques known in the art. These techniques areparticularly suitable, if the overexpressed gene encodes a cell surfaceprotein. Such binding assays are well known in the art and may beperformed essentially as described above.

In situ detection of antibody binding to the marker gene products can beperformed, for example, by immunofluorescence or immunoelectronmicroscopy. For this purpose, a histological specimen is removed fromthe patient, and a labeled antibody is applied to it, preferably byoverlaying the antibody on a biological sample. This procedure alsoallows for determining the distribution of the marker gene product inthe tissue examined. It will be apparent for those skilled in the artthat a wide variety of histological methods are readily available for insitu detection.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES

Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, 10801 University Boulevard, Manassas, Va.20110-2209.

Example 1 Isolation of cDNA Clones Encoding Human PRO301

The extracellular domain (ECD) sequences (including the secretion signalsequence, if any) from about 950 known secreted proteins from theSwiss-Prot public database were used to search EST databases. The ESTdatabases included public EST databases (e.g., GenBank), a proprietaryEST database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, Calif.). Thesearch was performed using the computer program BLAST or BLAST2[Altschul et al., Methods in Enzymology, 266:460-480 (1996)] as acomparison of the ECD protein sequences to a 6-frame translation of theEST sequences. Those comparisons resulting in a BLAST score of 70 (or insome cases, 90) or greater that did not encode known proteins wereclustered and assembled into consensus DNA sequences with the program“phrap” (Phil Green, University of Washington, Seattle, Wash.).

A consensus DNA sequence encoding DNA35936 was assembled using phrap. Insome cases, the consensus DNA sequence was extended using repeatedcycles of blast and phrap to extend the consensus sequence as far aspossible using the three sources of EST sequences listed above.

Based on this consensus sequence, oligonucleotides were synthesized: 1)to identify by PCR a cDNA library that contained the sequence ofinterest, and 2) for use as probes to isolate a clone of the full-lengthcoding sequence. Forward and reverse PCR primers (notated as *.f and*.r, respectively) may range from 20 to 30 nucleotides (typically about24), and are designed to give a PCR product of 100-1000 bp in length.The probe sequences (notated as *.p) are typically 40-55 bp (typicallyabout 50) in length. In some cases, additional oligonucleotides aresynthesized when the consensus sequence is greater than 1-1.5 kbp. Inorder to screen several libraries for a source of a full-length clone,DNA from the libraries was screened by PCR amplification, as per Ausubelet al., Current Protocols in Molecular Biology, with the PCR primerpair. A positive library was then used to isolate clones encoding thegene of interest by the in vivo cloning procedure suing the probeoligonucleotide and one of the PCR primers.

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO301 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalkidney. The cDNA libraries used to isolated the cDNA clones wereconstructed by standard methods using commercially available reagents(e.g., Invitrogen, San Diego, Calif.; Clontech, etc.) The cDNA wasprimed with oligo dT containing a NotI site, linked with blunt to SalIhemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D thatdoes not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991)) in the unique XhoI and NotI sites.

A cDNA clone was sequenced in its entirety. The full-length nucleotidesequence of native sequence DNA40628 is shown in FIG. 5 (SEQ ID NO: 11).Clone DNA40628 contains a single open reading frame with an apparenttranslational initiation site at nucleotide positions 52-54 (FIG. 5; SEQID NO: 11). The predicted polypeptide precursor is 299 amino acids longwith a predicted molecular weight of 32583 daltons and pI of 8.29. CloneDNA40628 has been deposited with ATCC and is assigned ATCC deposit No.209432.

Based on a BLAST and FastA sequence alignment analysis of thefull-length sequence, PRO301 encoded by DNA40628 shows amino acidsequence identity to A33 antigen precursor (30%) and coxsackie andadenovirus receptor protein (29%).

The oligonucleotide sequences used in the above procedure were thefollowing:

OLI2162 (35936.f1) (SEQ ID NO: 12) TCGCGGAGCTGTGTTCTGTTTCCC OLI2163(35936.p1) (SEQ ID NO: 13)TGATCGCGATGGGGACAAAGGCGCAAGCTCGAGAGGAAACTGTTGTGC CT OLI2164 (35936.f2)(SEQ ID NO: 14) ACACCTGGTTCAAAGATGGG OLI2165 (35936.r1) (SEQ ID NO: 15)TAGGAAGAGTTGCTGAAGGCACGG OLI2166 (35936.f3) (SEQ ID NO: 16)TTGCCTTACTCAGGTGCTAC OLI2167 (35936.r2) (SEQ ID NO: 17)ACTCAGCAGTGGTAGGAAAG

Example 2 Isolation of cDNA Clones Encoding Human PRO362

The extracellular domain (ECD) sequences (including the secretionsignal, if any) of about 950 known secreted proteins from the Swiss-Protpublic protein database were used to search expressed sequences tag(EST) databases. The EST databases included public EST databases (e.g.,GenBank) and a proprietary EST DNA database (LIFESEQ®, IncytePharmaceuticals, Palo Alto, Calif.). The search was performed using thecomputer program BLAST or BLAST-2 (e.g., Altshul et al., Methods inEnzymology 266: 460-480 (1996)) as a comparison of the ECD proteinsequences to a 6 frame translation of the EST sequence. Thosecomparisons resulting in a BLAST score 70 (or in some cases 90) orgreater that did not encode known proteins were clustered and assembledinto consensus DNA sequences with the program “phrap” (Phil Green,University of Washington, Seattle, Wash.

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap. This consensus sequence is herein designated DNA42257 (SEQID NO: 5) (see FIG. 4C). Based on the DNA42257 (SEQ ID NO: 5) consensussequence shown in FIG. 4C, oligonucleotides were synthesized: 1) toidentify by PCR a cDNA library that contained the sequence of interest,and 2) for use as probes to isolate a clone of the full-length codingsequence for PRO362. Forward and reverse PCR primers generally rangefrom 20 to 30 nucleotides and are often designed to give a PCR productof about 100-1000 bp in length. The probe sequences are typically 40-55bp in length. In some cases, additional oligonucleotides are synthesizedwhen the consensus sequence is greater than about 1-1.5 kbp. In order toscreen several libraries for a full-length clone, DNA from the librarieswas screened by PCR amplification, as per Ausubel et al., CurrentProtocols in Molecular Biology, with the PCR primer pair. A positivelibrary was then used to isolate clones encoding the gene of interestusing the probe oligonucleotide and one of the primer pairs.

PCR primers (forward and reverse) were synthesized: forward PCR primer 1(42257.f1) (SEQ ID NO: 18) 5′-TATCCCTCCAATTGAGCACCCTGG-3′ forward PCRprimer 2 (42257.f2) (SEQ ID NO: 19) 5′-GTCGGAAGACATCCCAACAAG-3′ reversePCR primer 1 (42257.r1) (SEQ ID NO: 20) 5′-CTTCACAATGTCGCTGTGCTGCTC-3′reverse PCR primer 2 (42257.r2 (SEQ ID NO: 21)5′-AGCCAAATCCAGCAGCTGGCTTAC-3′

Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA42257 sequence which had the followingnucleotide sequence:

Hybridization probe (42257.p1) (SEQ ID NO: 22)5′-TGGATGACCGGAGCCACTACACGTGTGAAGTCACCTGGCAGACTCCT GAT-3′.

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pairs identified above. A positive library was then used toisolate clones encoding the PRO362 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalbrain tissue (LIB153). The cDNA libraries used to isolate the cDNAclones were constructed by standard methods using commercially availablereagents such as those from Invitrogen, San Diego, Calif. The cDNA wasprimed with oligo dT containing a NotI site linked with blunt to SalIhemikinased adaptors, cleaved with NotI, sized appropriately be gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D thatdoes not contain the SfiI site; see Holmes et al., Science 253:1278-1280 (1991)) in the unique XhoI and NotI sites.

DNA sequencing of the clones isolated as described gave the full-lengthDNA sequence for an isolated PRO362 (herein designated as UNQ317(DNA45416-1251) (SEQ ID NO: 7).

The entire nucleotide sequence of UNQ317 (DNA45416-1251) is shown inFIG. 6 (SEQ ID NO: 7). Clone UNQ367 (DNA45416-1251) (SEQ ID NO: 7)contains a single open reading frame with an apparent translationalinitiation site at nucleotide positions 1082-1084 (FIG. 6, SEQ ID NO:7). The predicted polypeptide precursor is 321 amino acids long (FIG. 3,SEQ ID NO: 2). The full-length PRO362 protein shown if FIG. 3 has anestimated molecular weight of about 35,544 daltons and a pI of about8.51. Analysis of the full-length PRO362 polypeptide as shown in FIG. 3(SEQ ID NO: 2) evidences the presence of a glycosaminoglycan attachmentsite at about amino acid 149 to about amino acid 152 and a transmembranedomain from about amino acid 276 to about amino acid 306. Clone UNQ317(DNA45416-1251) has been deposited with ATCC deposit No.: 209620.

Example 3 Isolation of cDNA Clones Encoding Human PRO245

The extracellular domain (ECD) sequences (including the secretionsignal, if any) of about 950 known secreted proteins from the Swiss-Protpublic protein database were used to search expressed sequences tag(EST) databases. The EST databases included public EST databases (e.g.,GenBank) and a proprietary EST DNA database (LIFESEQ®, IncytePharmaceuticals, Palo Alto, Calif.). The search was performed using thecomputer program BLAST or BLAST-2 (e.g., Altshul et al., Methods inEnzymology 266: 460-480 (1996)) as a comparison of the ECD proteinsequences to a 6 frame translation of the EST sequence. Thosecomparisons resulting in a BLAST score 70 (or in some cases 90) orgreater that did not encode known proteins were clustered and assembledinto consensus DNA sequences with the program “phrap” (Phil Green,University of Washington, Seattle, Wash.

A consensus DNA sequence was assembled relative to other EST sequences,wherein the consensus sequence is herein designated DNA30954 (SEQ ID NO:27). Based on the DNA30954 consensus sequence, oligonucleotides weresynthesized to identify by PCR a cDNA library that contained thesequence of interest and for use as probes to isolate a clone of thefull-length coding sequence for PRO245.

A pair of PCR primers (forward and reverse) were synthesized:

(SEQ ID NO: 28) forward PCR primer 5′-ATCGTTGTGAAGTTAGTGCCCC-3′ (SEQ IDNO: 29) reverse PCR primer 5′-ACCTGCGATATCCAACAGAATTG-3′

Forward and reverse PCR primers generally range from 20 to 30nucleotides and are often designed to give a PCR product of about100-1000 bp in length. The probe sequences are typically 40-55 bp inlength. In some cases, additional oligonucleotides are synthesized whenthe consensus sequence is greater than about 1-1.5 kbp. In order toscreen several libraries for a full-length clone, DNA from the librarieswas screened by PCR amplification, as per Ausubel et al., CurrentProtocols in Molecular Biology, with the PCR primer pair.

Additionally, a synthetic oligonucleotide hybridization probes wasconstructed from the consensus DNA30954 sequences which had thefollowing nucleotide sequence:

hybridization probe: (SEQ ID NO: 30)5′-GGAAGAGGATACAGTCACTCTGGAAGTATTAGTGGCTCCAGCAGTT CC-3′.

In order to screen several libraries for a source of a full-lengthclone, DNA form the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolated clones encoding the PRO245 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalliver tissue. The cDNA libraries used to isolate the cDNA clones wereconstructed by standard methods using commercially available reagentssuch as those from Invitrogen, San Diego, Calif. The cDNA was primedwith oligo dT containing a NotI site, linked with blunt to SalIhemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D thatdoes not contain the SfiI site; see Holmes et al., Science 253:1278-1280 (1991)) in the unique XhoI and NotI sites.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for a native sequence PRO245 [herein designatedas UNQ219 (DNA35638) (SEQ ID NO: 8)] and the derived protein sequence(SEQ ID NO: 9).

The entire nucleotide sequence of UNQ219 (DNA35638) is shown in FIG. 7(SEQ ID NO: 8). Clone UNQ219 (DNA35638) (SEQ ID NO: 8) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 89-91 (Kozak et al., supra) and ending at the stopcodon at nucleotide positions 1025-1027 (FIG. 7, SEQ ID NO: 8). Thepredicted polypeptide precursor is 312 amino acids long (FIG. 11) (SEQID NO: 9). Clone UNQ219 (DNA35638) has been deposited with the ATCC onSep. 17, 1997 and is assigned ATCC deposit No. 209265.

Example 4 Inhibition of VEGF Stimulated Proliferation of EndothelialCell Growth

Bovine adrenal cortical capillary endothelial (ACE) cells (from primaryculture, maximum 12-14 passages) were plated on 96-well microtiterplates (Amersham Life Science) at a density of 500 cells/well per 100 μLin low glucose DMEM, 10% calf serum, 2 mM glutamine, 1× pen/strep andfungizone, supplemented with 3 ng/mL VEGF. Controls were plated the sameway but some did not include VEGF. A test sample of the PRO301 andPRO245 polypeptide was added in a 100 μl volume for a 200 mcL finalvolume. Cells were incubated for 6-7 days at 37° C. The media wasaspirated and the cells washed 1× with PBS. An acid phosphatase reactionmixture (100 μL, 0.1M sodium acetate, pH 5.5, 0.1% Triton-100, 10 mMp-nitrophenyl phosphate) was added. After incubation for 2 hours at 37°C., the reaction was stopped by addition of 10 mcL 1N NaOH. OD wasmeasured on microtiter plate reader at 405 nm. Controls were no cells,cells alone, cells+FGF (5 ng/mL), cells+VEGF (3 ng/mL), cells+VEGF (3ng/ml)+TGF-β (1 ng/ml), and cells+VEGF (3 ng/mL)+LIF (5 ng/mL). (TGF-βat a 1 ng/ml concentration is known to block 70-90% of VEGF stimulatedcell proliferation.)

The results were assessed by calculating the percentage inhibition ofVEGF (3 ng/ml) stimulated cells proliferation, determined by measuringacid phosphatase activity at OD₄₀₅ nm, (1) relative to cells withoutstimulation, and (2) relative to the reference TGF-β inhibition of VEGFstimulated activity. The results, shown in Table 1, are indicative ofthe utility of the PRO301 and PRO245 polypeptide in the inhibition ofcell growth, especially cancer therapy and specifically in inhibitingtumor angiogenesis.

TABLE 1 % Proliferation relative to Compound Tested Concentrationcontrol DNA40628 protein (SEQ ID NO: 1)  7.0 nM 1.02 DNA40628 protein(SEQ ID NO: 1) 70.0 nM 0.88 DNA40628 protein (SEQ ID NO: 1) 700.0 nM 0.44 DNA40628 protein (SEQ ID NO: 1) 0.01%  0.92 DNA40628 protein (SEQID NO: 1) 0.1% 0.85 DNA40628 protein (SEQ ID NO: 1) 1.0% 0.68 DNA35638protein (SEQ ID NO: 9) 0.01%  0.76 DNA35638 protein (SEQ ID NO: 9) 0.1%0.35 DNA35638 protein (SEQ ID NO: 9) 1.0% 0.11 DNA35638 protein (SEQ IDNO: 9) 0.48 nM 1.03 DNA35638 protein (SEQ ID NO: 9)  4.8 nM 0.95DNA35638 protein (SEQ ID NO: 9) 48.0 nM 0.49

Example 5 Stimulatory Activity in Mixed Lymphocyte Reaction (MLR) Assay

The following describes assays for determining whether PRO301, PRO362,PRO245 and PRO1868 polypeptides are able to stimulate proliferation ofstimulated T-lymphocytes. Compounds which stimulate proliferation oflymphocytes are useful therapeutically where enhancement of aninflammatory response is beneficial, for example enhancement of theimmune response against neoplasia. Antagonists to such compounds thatstimulate proliferation of lymphocytes are useful therapeutically wherea reduction in the inflammatory response is beneficial. A therapeuticagent may take the form of an agonist or an antagonist of thepolypeptide of the invention, for example, murine-human chimeric,humanized or human antibodies against the polypeptide.

The basic protocol for this assay is described in Current Protocol inImmunology, Unit 3.12, J. E. Coligan, A. M. Kruisbeek, D H Marglies, E MShevach and W Strober, Eds, National Institute of Health, Published byJohn Wiley & Sons, Inc.

More specifically, in one assay variant, peripheral blood mononuclearcells (PBMC) are isolated from mammalian individuals, for example ahuman volunteer, by leukopheresis (one donor will supply stimulatoryPBMCs, the other donor will supply responder PBMCs). If desired, thecells are frozen in fetal bovine serum and DMSO after isolation. Frozencells may be thawed overnight in assay media (37° C., 5% CO₂) and thenwashed and resuspended to 3×10⁶ cells/ml of assay media (RPMI; 10% fetalbovine serum, 1% penicillin/streptomycin, 1% glutamine, 1% HEPES, 1%non-essential amino acids, 1% pyruvate).

The stimulator PBMCs are prepared by irradiating the cells (about 3000Rads). The assay is prepared by plating in triplicate wells a mixtureof: 100 μl of test sample diluted to 1% or 0.1%; 501 of irradiatedstimulator cells and 50 of responder PBMC cells. 100 μL of cell culturemedia or 100 ml of CD4-IgG is used as the control. The wells are thenincubated at 37° C., 5% CO₂ for 4 days. On day 5, each well is pulsedwith tritiated thymidine (1.0 mC/well; Amersham). The cells are washed 3times and then the uptake of the label is evaluated.

PRO301, PRO362 and PRO245 polypeptides were tested in another variant ofthe assay. In this variant assay, PBMC's were isolated from the spleensof BALB/c mice and C57B6 mice. The cells were teased from freshlyharvested spleens in assay media (RPMI; 10% fetal bovine serum, 1%penicillin/streptomycin, 1% glutamine, 1% HEPES, 1% non-essential aminoacids, 1% pyruvate) and the PBMCs were isolated by overlaying thesecells over Lympholyte M (Organon Teknika), centrifuging at 2000 rpm for20 minutes, collecting and washing the mononuclear cell layer in assaymedia and resuspending the cells to 1×10⁷ cells/ml of assay media. Theassay was then conducted as described above.

The results, shown below in Table 2, indicate that the PRO301, PRO362and PRO245 polypeptides of the invention are active as stimulators ofthe proliferation of stimulated T-lymphocytes. Positive increases overcontrol are considered positive with increases of greater than or equalto 180% being preferred. However, any value greater than controlindicates a stimulatory effect for the test protein.

TABLE 2 Percent Increase Compound Concentration over Control DNA40628protein (SEQ ID NO: 1) 0.1% 181.7 DNA40628 protein (SEQ ID NO: 1) 1.0%187.3 DNA40628 protein (SEQ ID NO: 1) 0.1% 193.4 DNA40628 protein (SEQID NO: 1) 1.0% 204.1 DNA45416 protein (SEQ ID NO: 2) 0.1% 87.4 DNA45416protein (SEQ ID NO: 2) 1.0% 180.2 DNA35638 protein (SEQ ID NO: 9) 0.1%189.7 DNA35638 protein (SEQ ID NO: 9) 0.1% 193.7 DNA35638 protein (SEQID NO: 9) 1.0% 212.5 DNA35638 protein (SEQ ID NO: 9) 1.0% 300.5

Example 6 Inflammatory Cell Infiltrates into Guinea Pig Skin

The following example shows that the polypeptides of the invention areproinflammatory in that they stimulate inflammatory cell infiltrates(i.e., neutrophilic, eosinophilic, monocytic or lymphocytic) into guineapig skin. The assay described herein monitors the capacity of eachprotein to induce an inflammatory cell infiltrate into the skin of aguinea pig. Compounds which stimulate inflammatory infiltration areuseful therapeutically where enhancement of an inflammatory response isbeneficial. Compounds which inhibit proliferation of lymphocytes areuseful therapeutically where suppression of an inflammatory response isbeneficial. A therapeutic agent may take the form of antagonists of thepolypeptides of the invention, for example, murine-human chimeric,humanized or human antibodies against the polypeptide.

Hairless guinea pigs (Charles River Labs) weighing 350 grams or morewere anesthetized with ketamine (75-80 mg/kg body weight) and xylazine(5 mg/kg body weight) intramuscularly. The protein samples of PRO301,PRO362 and PRO245 and control proteins were injected intradermally intothe backs of each animal at a volume of 100 μl per injection site. Therewere approximately 16-24 injection sites per animal. One mL of Evansblue dye (1% in physiological buffered saline) was injectedintracardially. The animals were euthanized after 6 hours and each skininjection site was biopsied and fixed in formalin. The skins wereprepared for histopathological evaluation. Each site was evaluated forinflammatory cell infiltration into the skin. Sites with visibleinflammatory cells were scored as positive. Samples inducing aninflammatory cell infiltrate were scored as proinflammatory substances.

TABLE 3 Compound Proinflammatory activity DNA40628 protein (SEQ IDNO: 1) + DNA45416 protein (SEQ ID NO: 2) + DNA35638 protein (SEQ ID NO:9) + Negative control −

Based on these results PRO1868 (SEQ ID NO: 31) also likely hasproinflammatory activity.

Example 7 Interaction with Human Neutrophils

The following example shows the ability of the polypeptides of theinvention to bind to human neutrophils, a molecule associated withinflammation and the inflammatory response.

Neutrophils isolated from the blood of human donors (PMN) as describedin Scan. J. Clin. Lab Invest. Suppl. 97: 51-76 (1968), were incubatedwith an Ig-fusion of protein encoded by DNA40628 (prepared as discussedin the following examples) or a negative control humanized antibody.

The PMNs were resuspended in a microfuge tube in PBS at a density of2×10⁶ cell equivalents per condition. The cells were washed twice withice cold PBS and pelleted at 400×g between washes. The PMN cells wereblocked with 0.5% BSA in PBS (blocking reagent) at 4° C. for 1 hour.After the incubation, the cells were further washed two additional timeswith blocking reagent. The PMNs were pelleted after the final wash andresuspended in 1 ml of blocking buffer at 0.1 μg/ml in both DNA40628protein and control antibody. The incubation was carried out for 2 hoursat 4° C. The PMN cells were gently resuspended every 15 minutes on ice,then washed and pelleted 5 times in blocking buffer, with each washlasting 5 minutes at 4° C. and pelleting occurring at 400×g. A 1:1000dilution of goat and anti-human IgG Fc specific-alkalinephosphatase-conjugated in the blocking buffer was then applied to thePMN cells. The PMN cells were incubated for 1 hour at 4° C., with gentlymixing every 15 minutes on ice. The PMN cells were then washed 5 timeswith blocking buffer, resuspended in the appropriate substrate foralkaline phosphatase and distributed in 4 equi-100 μl aliquots onto amicrotiter plate. Color development was read at O.D. 405. The resultsare shown in FIG. 21.

Example 8 Dot Blot Tissue Hybridization

A human RNA master blot (Clontech) was hybridized overnight at 65° C. inEXPRESSHYB® buffer (Clontech) per the manufacturer's instructions with100 nM of psoralen-biotin labeled DNA40628 cDNA probe (SEQ ID NO: 7).Streptavidin-alkaline phosphatase was used to detect the biotinylatedprobe. The blot was developed with CDP-star substrate (Ambion) andexposed for various times on Biomax film (Kodak). A cDNA hybridizationanalysis of human tissues show that DNA40628 mRNA is expressed in a widerange of tissues, but not in the cerebellum and spinal cord (FIG. 19).DNA40628 mRNA is highly expressed in the colon, prostate, stomach,ovary, salivary gland, kidney, lung, trachea and placenta.

Example 9 Gene Product Overexpression

This example shows that genes encoding the various proteins indicated inFIG. 20 are overexpressed in colitic colon of CRF2-4−/− “knock out”mice. Therapeutic agents may take the form of antagonists of theindicated gene products, for example, murine-human chimeric, humanizedor human antibodies thereagainst.

CRF 2-4−/− mice (Spencer et al., J. Exp. Med. 187, 571-578 (1998)), areIL-10 receptor knockout animals that have a subunit of the gene encodingthe IL-10 receptor removed. The mice are unresponsive to thedownregulatory functions of IL-10 for macrophage activation, and cannotdownregulate response to lipopolysaccharide triggering of macrophageTNF-α secretion. They develop a chronic colitis which can lead tocolonic adenocarcinoma. The spontaneous colitis is mediated bylymphocytes, monocytes and neutrophils. IL-10 suppresses theinflammatory response by modulating expression of certain inflammatorycytokines.

The probes for the proteins indicated in FIG. 20 were created from mRNAtemplates for the indicated gene products and used in the 5′-nucleaseassay (e.g., TAQMAN™) and real-time quantitative PCR (e.g., ABI PRIZM7700 SEQUENCE DETECTION SYSTEM™ (Perkin-Elmer, Applied BiosystemsDivision, Foster City, Calif.). The results are reported in delta CTunits. One unit corresponds to 1 PCR cycle or approximately a 2-foldamplification relative to normal, two units correspond to 4-fold, 3units to 8-fold, etc. Quantitation was obtained using primers and aTAQMAN™ fluorescent tagged-mRNA derived from the testedinflammatory-related gene products indicated in FIG. 20. Regions of theindicated gene products which are most likely to contain unique nucleicacid sequences and which are least likely to have spliced out intronsare preferred for the primer derivation, e.g. 3′-untranslated region.

The 5′-nuclease assay reaction is a fluorescent PCR-based techniquewhich makes use of the 5′-exonuclease activity of Taq DNA polymeraseenzyme to monitor amplification in real time. Two oligonucleotideprimers are used to generate an amplicon typical of a PCR reaction. Athird oligonucleotide, or probe, is designed to detect nucleotidesequence located between the two PCR primers. The probe isnon-extendible by Taq DNA polymerase enzyme, and is labeled with areported fluorescent dye and a quencher fluorescent dye. Anylaser-induced emission from the reporter dye is quenched by thequenching dye when the two dyes are located close together as they areon the probe. During the amplification reaction, the probe is cleaved bythe Taq DNA polymerase enzyme in a template-dependent manner. Theresultant probe fragments disassociate in solution, and the signal fromthe release reporter dye is free from the quenching effect of the secondfluorophore. One molecule of reporter dye is liberated for each newmolecule synthesized, and detection of the unquenched reporter dyeprovided the basis for quantitative interpretation of the data.

The 5′-nuclease procedure is run on a real-time quantitative PCR devicesuch as the ABI Prism 7700™ Sequence Detection. The system consists of athermocycler, laser, charge-coupled device (CCD) camera and computer.The system amplifies samples in a 96-well format on a thermocycler.During amplification, laser-induced fluorescent signal is collected inreal-time through fiber optics cables for all 96 wells, and detected atthe CCD. The system includes software for running the instrument and foranalyzing the data.

The 5′-nuclease assay data are initially expressed as Ct, or thethreshold cycle. This is defined as the cycle at which the reportersignal accumulates above the background level of fluorescence. The Ctvalues are used as quantitative measurement of the relative number ofstarting copies of a particular target sequence in a nucleic acidsample.

The results of the mRNA amplification are shown in FIG. 20. Expressionin wild-type animals were compared with CRF2-4−/− KO animals withbeta-actin as the reference standard. Four animals were measured in eachgroup. All four KO animals were diagnosed with colitis and in addition,three of these had colon adenocarcinoma.

FIG. 20 shows that JAM mRNA is increased 3.3-fold in the colon ofCRF2-4−/− mice with colitis.

As a result, it is likely that PRO301, PRO362, PRO245 and PRO1868 wouldalso have elevated expression in inflammatory human disease, such asinflammatory bowel disease and other inflammatory diseases of the gut.

Example 10 Induction of Endothelial Cell Apoptosis

The ability of the polypeptides of the invention to induce apoptosis inendothelial cells was tested in human venous umbilical vein endothelialcells (HUVEC, Cell Systems). The first day, the cells were plated on96-well microtiter plates (Amersham Life Sciences, cytostar-Tscintillating microplate, RPNQ160, sterile, tissue-culture treated,individually wrapped), in 10% serum (CSG-medium, Cell Systems), at adensity of 2×10⁴ cells per well in a total volume of 100 μl. The secondday, PRO301 and PRO245 polypeptide encoded by DNA40628 and DNA35638,respectively, was added in triplicate at dilutions of 1%, 0.33% and0.11%. On the third day, the ability of the PRO301 and PRO245polypeptides to induce apoptosis was determined using a commerciallyavailable kit, Apoptosis Detection Kit (R&D Systems, Minnesota) in whichannexin V, a member of the calcium and phospholipid binding proteins, isused to detect apoptosis, following the protocol recommended by themanufacturer. Fluroescein-labeled annexin V and propidium iodide wereadded to the cells. Analysis was performed with cytometers equipped witha single laser emitting excitation light at 488 nm. In this test, livecells will not stain with either fluorochrome, necrotic cells will stainwith both fluorochromes, and cells undergoing apoptosis will stain onlywith the annexin V-FITC reagent. The annexin V-FITC generated signal wasdetected in the FITC signal detector. The results are indicated in theTable 4 below.

TABLE 4 % over background Compound tested Concentration fluorescenceDNA40628 protein (SEQ ID NO: 1) 0.11% 115.8 DNA40628 protein (SEQ IDNO: 1) 0.33% 199.3 DNA40628 protein (SEQ ID NO: 1)  1.0% 335.6 DNA35638protein (SEQ ID NO: 9) 0.11% 77.6 DNA35638 protein (SEQ ID NO: 9) 0.33%143.7 DNA35638 protein (SEQ ID NO: 9)  1.0% 146.0 DNA35638 protein (SEQID NO: 9) 6.82 nM 67.2 DNA35638 protein (SEQ ID NO: 9) 20.46 nM  102.6DNA35638 protein (SEQ ID NO: 9) 62.0 nM 118.8

The ability of the protein compounds of the invention to induceendothelial cell apoptosis, particularly in combination with thedisruption of cell junction formation as indicated in Example 4 isindicative that the compounds play roles in cell adhesion andtransmigration. Similar to murine JAM, the compounds are likely celljunction molecules in epithelia and endothelia, which explains theirbroad tissue distribution. The induction of endothelial cell apoptosisindicates a role in cell growth and apoptosis.

Example 11 In Vitro Antitumor Assay

The antiproliferative activity of the PRO301 and PRO362 polypeptides ofthe invention was determined in the investigational, disease-oriented invitro anti-cancer drug discovery assay of the National Cancer Institute(NCI), using sulforhodamine B (SRB) dye binding assay essentially asdescribed by Skehan et al., J. Natl. Cancer Inst. 82: 1107-1112 (1990).The 60 tumor cell lines employed in this study (“the NCI panel”) as wellas conditions for their maintenance and culture in vitro have beendescribed by Monks et al., J. Natl. Cancer Inst. 83: 757-766 (1991). Thepurpose of this screen is to initially evaluate the cytotoxic and/orcytostatic activity of the test compounds against different types oftumors (Monks et al., supra, Boyd, Cancer: Princ. Pract. Oncol. Update3(10): 1-12 (1989)).

Cells from the approximately 60 human tumor cell lines were harvestedwith trypsin/EDTA (Gibco), washed once, resuspended in IMEM and theirviability was determined. The cell suspensions were added by pipet (100μL volume) into separate 96-well microtiter plates. The cell density forthe 6-day incubation was less than for the 2-day incubation to preventovergrowth. Inoculates were allowed a preincubation period of 24 hoursat 37° C. for stabilization. Dilutions at twice the intended testconcentration were added at time zero in 100 ml aliquots to themicrotiter plates wells (1:2 dilution). Test compounds were evaluated atgiven half-log dilutions (1000 to 100,000 fold). Incubations took placefor two days and six days in a 5% CO₂ atmosphere and 100% humidity.

After incubation, the medium was removed and the cells were fixed in 0.1ml of 10% trichloroacetic acid at 40° C. The plates were rinsed fivetimes with deionized water, dried, stained for 30 minutes with 0.1 ml of0.4% sulforhodamine B dye (Sigma) dissolved in 1% acetic acid, rinsedfour times with 1% acetic acid to remove unbound dye, dried, and thestain was extracted for five minutes with 0.1 ml of 10 mM Tris base[tris(hydroxymethyl)aminomethane], pH 10.5. The absorbance (OD) ofsulforhodamine B at 492 nm was measured using a computer-interfaced,96-well microtiter plate reader.

A test sample was considered positive if it showed at least 50% growthinhibitory effect at one or more concentrations. The positive resultsare shown in the following table, where the abbreviations are asfollows:

NSCL=non-small lung carcinoma

CNS=central nervous system

Leuk=leukemia

TABLE 5 Length of Tumor cell line Test compound Concentration assay TypeDesignation DNA40628 0.075 nM   6 Colon HCC-2998 protein Melanoma M14(SEQ ID NO: 1) DNA40638 700 nM 6 Melanoma M14 protein (SEQ ID NO: 1)DNA40628 152 nM 6 Colon SR protein Melanoma LOX IMVI (SEQ ID NO: 1)DNA40628 15.2 nM  6 Melanoma LOX IMVI protein (SEQ ID NO: 1) DNA406280.85 nM  6 NSCL HOP62 protein Ovarian OVCAR-3 (SEQ ID NO: 1) ProstatePC3 DNA45416  15 nM 2 Ovarian SK-OV-3 protein (SEQ ID NO: 2) DNA45416 15 nM 6 NSCL NCI-H322M protein Prostate PC-3 (SEQ ID NO: 2) DNA45416 4.7 nM 6 Melanoma LOX IMVI protein (SEQ ID NO: 2) DNA45416  47 nM 6NSCL NCI-H322M protein Colon Colo 205 (SEQ ID NO: 2) DNA45416 152 nM 2CNS SR-295 protein Breast T047D (SEQ ID NO: 2) DNA45416 152 nM 6 LeukSR, HL-60 (TB), protein MOLT-4, K-562 (SEQ ID NO: 2) NSCL NCI-H23, EKVXColon HCC-2998 CNS U251 Melanoma UACC-62, UACC-257, LOX IMVI DNA356380.35 nM  2 NSCL HOP92 protein Ovarian OVCAR-4 (SEQ ID NO: 9) DNA356380.35 nM  2 Leuk SR protein (SEQ ID NO: 9) DNA35638 0.35 nM  6 ColonHCC-2998 protein (SEQ ID NO: 9) DNA35638  3.5 nM 6 Leuk SR protein ColonSW-620 (SEQ ID NO: 9) DNA35638  6.2 nM 6 Colon HCT-116 protein (SEQ IDNO: 9) DNA35638  6.2 nM 6 Leuk RPMI-8226 protein (SEQ ID NO: 9)

Example 12 Use of PRO301, PRO362, PRO245 or PRO1868 as a HybridizationProbe

The following method describes use of a nucleotide sequence encoding aPRO301, PRO362, PRO245 or PRO1868 as a hybridization probe.

DNA comprising the coding sequence of native sequence PRO301, PRO362,PRO245 or PRO1868 (as shown in FIGS. 5-7 and 61, SEQ ID NO: 11, 7, 8 and31), respectively, is employed as a probe to screen for homologous DNAs(such as those encoding naturally-occurring variants of PRO301, PRO362,PRO245 or PRO1868, respectively) in human tissue cDNA libraries or humantissue genomic libraries.

Hybridization and washing of filters containing either cDNA or genomiclibrary DNAs is performed under the following high stringencyconditions. Hybridization of radiolabeled PRO301-, PRO362-, PRO245 orPRO1868-derived probe to the filters is performed in a solution of 50%formamide, 5×SSC, 0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodiumphosphate, pH 6.8, 2×Denhardt's solution, and 10% dextran sulfate at 42°C. for 20 hours. Washing of the filters is performed in an aqueoussolution of 0.1×SSC and 0.1% SDS at 42° C.

DNAs having a desired sequence identity with the DNA encoding afull-length native sequence PRO301, PRO362, PRO245 or PRO1868 are thenidentified using standard techniques known in the art.

Example 13 Expression of PRO301, PRO362, PRO245 or PRO1868 in E. coli

This example illustrates preparation of an unglycosylated form ofPRO301, PRO362, PRO245 or PRO1868 by recombinant expression in E. coli.

The DNA sequence encoding PRO301, PRO362, PRO245 or PRO1868 is initiallyamplified using selected PCR primers. The primers should containrestriction enzyme sites which correspond to the restriction enzymesites on the selected expression vector. A variety of expression vectorsmay be employed. An example of a suitable vector is pBR322 (derived fromE. coli; see Bolivar et al., Gene, 2:95 (1977)) which contains genes forampicillin and tetracycline resistance. The vector is digested withrestriction enzyme and dephosphorylated. The PCR amplified sequences arethen ligated into the vector. The vector will preferably includesequences which encode for an antibiotic resistance gene, a trppromoter, a polyhis leader (including the first six STII codons, polyhissequence, and enterokinase cleavage site), the PRO301, PRO362, PRO245 orPRO1868 coding region, lambda transcriptional terminator, and an argUgene.

The ligation mixture is then used to transform a selected E. coli strainusing the methods described in Sambrook et al., supra. Transformants areidentified by their ability to grow on LB plates and antibioticresistant colonies are then selected. Plasmid DNA can be isolated andconfirmed by restriction analysis and DNA sequencing.

Selected clones are grown overnight in liquid culture medium such as LBbroth supplemented with antibiotics. The overnight culture issubsequently be used to inoculate a larger scale culture. The cells arethen grown to a desired optical density, during which the expressionpromoter is turned on.

After culturing the cells for several more hours, the cells areharvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized PRO301, PRO362, PRO245 or PRO1868 protein ispurified, for example by using a metal chelating column under conditionsthat allow tight binding of the protein.

PRO301 was expressed in E. coli in a poly-His tagged form, using thefollowing procedure. The DNA encoding PRO301 was initially amplifiedusing selected PCR primers. The primers contained restriction enzymesites which correspond to the restriction enzyme sites on the selectedexpression vector, and other useful sequences providing for efficientand reliable translation initiation, rapid purification on a metalchelation column, and proteolytic removal with enterokinase. ThePCR-amplified, poly-His tagged sequences were then ligated into anexpression vector, which was used to transform an E. coli host based onstrain 52 (W3110 fuhA(tonA) lon galE rpoHts(htpRts) clpP(lacIq).Transformants were first grown in LB containing 50 mg/ml carbenicillinat 30° C. with shaking until an O.D. 600 of 3-5 was reached. Cultureswere then diluted 50-100 fold into CRAP media (prepared by mixing 3.57 g(NH₄)₂SO₄, 0.71 g sodium citrateA2H2O, 1.07 g KCl, 5.36 g Difco yeastextract, 5.36 g Sheffield hycase SF in 500 mL water, as well as 110 mMMPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgSO₄) and grown forapproximately 20-30 hours at 30° C. with shaking. Samples were removedto verify expression by SDS-PAGE analysis, and the bulk culture iscentrifuged to pellet the cells. Cell pellets were frozen untilpurification and refolding.

E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) wasresuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8buffer. Solid sodium sulfite and sodium tetrathionate is added to makefinal concentrations of 0.1M and 0.02 M, respectively, and the solutionwas stirred overnight at 4° C. This step results in a denatured proteinwith all cysteine residues blocked by sulfitolization. The solution wascentrifuged at 40,000 rpm in a Beckman Ultracentrifuge for 30 min. Thesupernatant was diluted with 3-5 volumes of metal chelate column buffer(6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micronfilters to clarify. Depending the clarified extract was loaded onto a 5ml Qiagen Ni-NTA metal chelate column equilibrated in the metal chelatecolumn buffer. The column was washed with additional buffer containing50 mM imidazole (Calbiochem, Utrol grade), pH 7.4. The protein waseluted with buffer containing 250 mM imidazole. Fractions containing thedesired protein were pooled and stored at 4° C. Protein concentrationwas estimated by its absorbance at 280 nm using the calculatedextinction coefficient based on its amino acid sequence.

The protein was refolded by diluting sample slowly into freshly preparedrefolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3 M NaCl, 2.5 Murea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. Refolding volumes werechosen so that the final protein concentration was between 50 to 100micrograms/ml. The refolding solution was stirred gently at 4° C. for12-36 hours. The refolding reaction was quenched by the addition of TFAto a final concentration of 0.4% (pH of approximately 3). Before furtherpurification of the protein, the solution was filtered through a 0.22micron filter and acetonitrile was added to 2-10% final concentration.The refolded protein was chromatographed on a Poros R1/H reversed phasecolumn using a mobile buffer of 0.1% TFA with elution with a gradient ofacetonitrile from 10 to 80%. Aliquots of fractions with A280 absorbancewere analyzed on SDS polyacrylamide gels and fractions containinghomogeneous refolded protein were pooled. Generally, the properlyrefolded species of most proteins are eluted at the lowestconcentrations of acetonitrile since those species are the most compactwith their hydrophobic interiors shielded from interaction with thereversed phase resin. Aggregated species are usually eluted at higheracetonitrile concentrations. In addition to resolving misfolded forms ofproteins from the desired form, the reversed phase step also removesendotoxin from the samples.

Fractions containing the desired folded PRO301 protein, respectively,were pooled and the acetonitrile removed using a gentle stream ofnitrogen directed at the solution. Proteins were formulated into 20 mMHepes, pH 6.8 with 0.14 M sodium chloride and 4% mannitol by dialysis orby gel filtration using G25 Superfine (Pharmacia) resins equilibrated inthe formulation buffer and sterile filtered.

Example 14 Expression of PRO301, PRO362, PRO245 or PRO1868 in MammalianCells

This example illustrates preparation of a glycosylated form of a PRO301,PRO362, PRO245 Or PRO1868 by recombinant expression in mammalian cells.

The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), is employedas the expression vector. Optionally, the PRO301, PRO362, PRO245 orPRO186 DNA is ligated into pRK5 with selected restriction enzymes toallow insertion of the PRO301, PRO362, PRO245 or PRO1868 DNA usingligation methods such as described in Sambrook et al., supra. Theresulting vector is called pRK5-PRO301, pRK5-PRO362, pRK5-PRO245 orpRK5-PRO1868, respectively.

In one embodiment, the selected host cells may be 293 cells. Human 293cells (ATCC CCL 1573) are grown to confluence in tissue culture platesin medium such as DMEM supplemented with fetal calf serum andoptionally, nutrient components and/or antibiotics. About 10 μgpRK5-PRO301, pRK5-PRO362, pRK5-PRO245 DNA or pRK5-PRO1868 is mixed withabout 1 μg DNA encoding the VA RNA gene [Thimmappaya et al., Cell,31:543 (1982)] and dissolved in 500 μl of 1 mM Tris-HCl, 0.1 mM EDTA,0.227 M CaCl₂. To this mixture is added, dropwise, 500 μl of 50 mM HEPES(pH 7.35), 280 mM NaCl, 1.5 mM NaPO₄, and a precipitate is allowed toform for 10 minutes at 25° C. The precipitate is suspended and added tothe 293 cells and allowed to settle for about four hours at 37° C. Theculture medium is aspirated off and 2 ml of 20% glycerol in PBS is addedfor 30 seconds. The 293 cells are then washed with serum free medium,fresh medium is added and the cells are incubated for about 5 days.

Approximately 24 hours after the transfections, the culture medium isremoved and replaced with culture medium (alone) or culture mediumcontaining 200 μCi/ml ³⁵S-cysteine and 200 μCi/ml ³⁵S-methionine. Aftera 12 hour incubation, the conditioned medium is collected, concentratedon a spin filter, and loaded onto a 15% SDS gel. The processed gel maybe dried and exposed to film for a selected period of time to reveal thepresence of PRO301, PRO362, PRO245 or PRO1868 polypeptide. The culturescontaining transfected cells may undergo further incubation (in serumfree medium) and the medium is tested in selected bioassays.

In an alternative technique, PRO301, PRO362, PRO245 or PRO1868 DNA maybe introduced into 293 cells transiently using the dextran sulfatemethod described by Somparyrac et al., Proc. Natl. Acad. Sci, 12:7575(1981). 293 cells are grown to maximal density in a spinner flask and700 μg pRK5-PRO301, pRK5-PRO362, pRK5-PRO245 or pRK5-PRO1868 DNA isadded. The cells are first concentrated from the spinner flask bycentrifugation and washed with PBS. The DNA-dextran precipitate isincubated on the cell pellet for four hours. The cells are treated with20% glycerol for 90 seconds, washed with tissue culture medium, andre-introduced into the spinner flask containing tissue culture medium, 5μg/ml bovine insulin and 0.1 μg/ml bovine transferrin. After about fourdays, the conditioned media is centrifuged and filtered to remove cellsand debris. The sample containing expressed PRO301, PRO362, PRO245 orPRO1868 can then be concentrated and purified by any selected method,such as dialysis and/or column chromatography.

In another embodiment, PRO301, PRO362, PRO245 or PRO1868 can beexpressed in CHO cells. The pRK5-PRO301, pRK5-PRO362, pRK5-PRO245 orpRK5-PRO1868 can be transfected into CHO cells using known reagents suchas CaPO₄ or DEAE-dextran. As described above, the cell cultures can beincubated, and the medium replaced with culture medium (alone) or mediumcontaining a radiolabel such as ³⁵S-methionine. After determining thepresence of PRO301, PRO362, PRO245 or PRO1868 polypeptide, the culturemedium may be replaced with serum free medium. Preferably, the culturesare incubated for about 6 days, and then the conditioned medium isharvested. The medium containing the expressed PRO301, PRO362, PRO245 orPRO1868 can then be concentrated and purified by any selected method.

Epitope-tagged PRO301, PRO362, PRO245 or PRO1868 may also be expressedin host CHO cells. The PRO301, PRO362, PRO245 or PRO1868 may besubcloned out of the pRK5 vector. The subclone insert can undergo PCR tofuse in frame with a selected epitope tag such as a poly-his tag into aBaculovirus expression vector. The poly-his tagged PRO301, PRO362,PRO245 or PRO1868 insert can then be subcloned into a SV40 driven vectorcontaining a selection marker such as DHFR for selection of stableclones. Finally, the CHO cells can be transfected (as described above)with the SV40 driven vector. Labeling may be performed, as describedabove, to verify expression. The culture medium containing the expressedpoly-His tagged PRO301, PRO362, PRO245 or PRO1868 can then beconcentrated and purified by any selected method, such as byNi²⁺-chelate affinity chromatography.

PRO301, PRO362, PRO245 and PRO1868 were expressed in CHO cells by both atransient and stable expression procedure.

Stable expression in CHO cells was performed using the followingprocedure. The proteins were expressed as an IgG construct(immunoadhesin), in which the coding sequences for the soluble forms(e.g. extracellular domains) of the respective proteins were fused to anIgG1 constant region sequence containing the hinge, CH2 and CH2 domainsand/or as a poly-His tagged form.

Following PCR amplification, the respective DNAs were subcloned in a CHOexpression vector using standard techniques as described in Ausubel etal., Current Protocols of Molecular Biology, Unit 3.16, John Wiley andSons (1997). CHO expression vectors are generally constructed to havecompatible restriction sites 5= and 3= of the DNA of interest to allowthe convenient shuttling of cDNAs. The vector used here for expressionin CHO cells is as described in Lucas et al., Nucl. Acids Res. 24: 9(1774-1779 (1996), and uses the SV40 early promoter/enhancer to driveexpression of the cDNA of interest and dihydrofolate reductase (DHFR).DHFR expression permits selection for stable maintenance of the plasmidfollowing transfection.

Twelve micrograms of the desired plasmid DNA were introduced intoapproximately 10 million CHO cells using the commercially availabletransfection reagent Superfect⁷ (Qiagen), Dosper⁷ or Fugene⁷ (BoehringerMannheim). The cells were grown as described in Lucas et al., supra.Approximately 3×10⁻⁷ cells were frozen in an ampule for further growthand production as described below.

The ampules containing the plasmid DNA were thawed by placement into awater bath and mixed by vortexing. The contents were pipetted into acentrifuge tube containing 10 mLs of media and centrifuged at 1000 rpmfor 5 minutes. The supernatant was aspirated and the cells wereresuspended in 10 mL of selective media (0.2: m filtered PS20 with 5%0.2: m diafiltered fetal bovine serum). The cells were then aliquotedinto a 100 mL spinner containing 90 mL of selective media. After 1-2days, the cells were transferred into a 250 mL spinner filled with 150mL selective growth medium and incubated at 37EC. After another 2-3days, a 250 mL, 500 mL and 2000 mL spinners were seeded with 3×10⁵cells/mL. The cell media was exchanged with fresh media bycentrifugation and resuspension in production medium. Although anysuitable CHO media may be employed, a production medium described inU.S. Pat. No. 5,122,469, issued Jun. 16, 1992 was actually used. 3 Lproduction spinner is seeded at 1.2×10⁶ cells/mL. On day 0, the cellnumber and pH were determined. On day 1, the spinner was sampled andsparging with filtered air was commenced. On day 2, the spinner wassampled, the temperature shifted to 33EC, and 30 mL of 500 g/L glucoseand 0.6 mL of 10% antifoam (e.g., 35% polydimethylsiloxane emulsion, DowCorning 365 Medical Grade Emulsion). Throughout the production, pH wasadjusted as necessary to keep at around 7.2. After 10 days, or whenviability dropped below 70%, the cell culture was harvested bycentrifugation and filtering through a 0.22: m filter. The filtrate waseither stored at 4EC or immediately loaded onto columns forpurification.

For the poly-His tagged constructs, the proteins were purified using aNi-NTA column (Qiagen). Before purification, imidazole was added to theconditioned media to a concentration of 5 mM. The conditioned media waspumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4,buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5ml/min. at 4EC. After loading, the column was washed with additionalequilibration buffer and the protein eluted with equilibration buffercontaining 0.25 M imidazole. The highly purified protein wassubsequently desalted into a storage buffer containing 10 mM Hepes, 0.14M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia)column and stored at −80EC.

Immunoadhesin (Fc containing) constructs of the proteins were purifiedfrom the conditioned media as follows. The conditioned medium was pumpedonto a 5 ml Protein A column (Pharmacia) which had been equilibrated in20 mM Na phosphate buffer, pH 6.8. After loading, the column was washedextensively with equilibration buffer before elution with 100 mM citricacid, pH 3.5. The eluted protein was immediately neutralized bycollecting 1 ml fractions into tubes containing 275: L of 1 M Trisbuffer, pH 9. The highly purified protein was subsequently desalted intostorage buffer as described above for the poly-His tagged proteins. Thehomogeneity was assessed by SDS polyacrylamide gels and by N-terminalamino acid sequencing by Edman degradation.

PRO301, PRO362 PRO245 and PRO1868 were also produced by transientexpression in COS cells.

Example 15 Expression of PRO301, PRO362, PRO245 or PRO1868 in Yeast

The following method describes recombinant expression of PRO301, PRO362,PRO245 or PRO1868 in yeast.

First, yeast expression vectors are constructed for intracellularproduction or secretion of PRO301, PRO362, PRO245 or PRO1868 from theADH2/GAPDH promoter. DNA encoding PRO301, PRO362, PRO245 or PRO1868, aselected signal peptide and the promoter is inserted into suitablerestriction enzyme sites in the selected plasmid to direct intracellularexpression of PRO301, PRO362, PRO245 or PRO1868. For secretion, DNAencoding PRO301, PRO362, PRO245 or PRO1868 can be cloned into theselected plasmid, together with DNA encoding the ADH2/GAPDH promoter,the yeast alpha-factor secretory signal/leader sequence, and linkersequences (if needed) for expression of PRO301, PRO362, PRO245 orPRO1868.

Yeast cells, such as yeast strain AB110, can then be transformed withthe expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10% trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

Recombinant PRO301, PRO362, PRO245 or PRO1868 can subsequently beisolated and purified by removing the yeast cells from the fermentationmedium by centrifugation and then concentrating the medium usingselected cartridge filters. The concentrate containing PRO301, PRO362,PRO245 or PRO1868 may further be purified using selected columnchromatography resins.

Example 16 Expression of PRO301, PRO362, PRO245 or PRO1868 inBaculovirus-Infected Insect Cells

The following method describes recombinant expression of PRO301, PRO362or PRO245 in Baculovirus-infected insect cells.

The PRO301, PRO362, PRO245 or PRO1868 is fused upstream of an epitopetag contained with a baculovirus expression vector. Such epitope tagsinclude poly-his tags and immunoglobulin tags (like Fc regions of IgG).A variety of plasmids may be employed, including plasmids derived fromcommercially available plasmids such as pVL1393 (Novagen). Briefly, thePRO301, PRO362, PRO245 or PRO1868 or the desired portion of the PRO301,PRO362, PRO245 or PRO1868 (such as the sequence encoding theextracellular domain) is amplified by PCR with primers complementary tothe 5′ and 3′ regions. The 5′ primer may incorporate flanking (selected)restriction enzyme sites. The product is then digested with thoseselected restriction enzymes and subcloned into the expression vector.

Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BACULOGOLD™ virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4-5 days of incubation at 28° C., thereleased viruses are harvested and used for further amplifications.Viral infection and protein expression is performed as described byO'Reilley et al., Baculovirus expression vectors: A laboratory Manual,Oxford: Oxford University Press (1994).

Expressed poly-his tagged PRO301, PRO362, PRO245 or PRO1868 can then bepurified, for example, by Ni²⁺-chelate affinity chromatography asfollows. Extracts are prepared from recombinant virus-infected Sf9 cellsas described by Rupert et al., Nature, 362:175-179 (1993). Briefly, Sf9cells are washed, resuspended in sonication buffer (25 mL Hepes, pH 7.9;12.5 mM MgCl₂; 0.1 mM EDTA; 10% Glycerol; 0.1% NP-40; 0.4 M KCl), andsonicated twice for 20 seconds on ice. The sonicates are cleared bycentrifugation, and the supernatant is diluted 50-fold in loading buffer(50 mM phosphate, 300 mM NaCl, 10% Glycerol, pH 7.8) and filteredthrough a 0.45 Fm filter. A Ni²⁺-NTA agarose column (commerciallyavailable from Qiagen) is prepared with a bed volume of 5 mL, washedwith 25 mL of water and equilibrated with 25 mL of loading buffer. Thefiltered cell extract is loaded onto the column at 0.5 mL per minute.The column is washed to baseline A₂₈₀ with loading buffer, at whichpoint fraction collection is started. Next, the column is washed with asecondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% Glycerol, pH6.0), which elutes nonspecifically bound protein. After reaching A₂₈₀baseline again, the column is developed with a 0 to 500 mM Imidazolegradient in the secondary wash buffer. One mL fractions are collectedand analyzed by SDS-PAGE and silver staining or western blot withNi²⁺-NTA-conjugated to alkaline phosphatase (Qiagen). Fractionscontaining the eluted His₁₀-tagged PRO301, PRO362, PRO245 or PRO1868 arepooled and dialyzed against loading buffer.

Alternatively, purification of the IgG tagged (or Fc tagged) PRO301,PRO362, PRO245 or PRO1868 can be performed using known chromatographytechniques, including for instance, Protein A or protein G columnchromatography.

PRO301, PRO362 and PRO245 were expressed in baculovirus infected Sf9insect cells. While the expression was actually performed in a 0.5-2 Lscale, it can be readily scaled up for larger (e.g. 8 L) preparations.The proteins were expressed as an IgG construct (immunoadhesin), inwhich the protein extracellular region was fused to an IgG1 constantregion sequence containing the hinge, CH2 and CH3 domains and/or inpoly-His tagged forms.

Following PCR amplification, the respective coding sequences weresubcloned into a baculovirus expression vector (pb.PH.IgG for IgGfusions and pb.PH.His.c for poly-His tagged proteins), and the vectorand BACULOGOLD™ baculovirus DNA (Pharmingen) were co-transfected into105 Spodoptera frugiperda (“Sf9”) cells (ATCC CRL 1711), usingLipofectin (Gibco BRL). pb.PH.IgG and pb.PH.His are modifications of thecommercially available baculovirus expression vector pVL1393(Pharmingen), with modified polylinker regions to include the His or Fctag sequences. The cells were grown in Hink's TNM-FH medium supplementedwith 10% FBS (Hyclone). Cells were incubated for 5 days at 28° C. Thesupernatant was harvested and subsequently used for the first viralamplification by infecting Sf9 cells in Hink's TNM-FH mediumsupplemented with 10% FBS at an approximate multiplicity of infection(MOI) of 10. Cells were incubated for 3 days at 28° C. The supernatantwas harvested and the expression of the constructs in the baculovirusexpression vector was determined by batch binding of 1 ml of supernatantto 25 mL of Ni-NTA beads (QIAGEN) for histidine tagged proteins orProtein-A Sepharose CL-4B beads (Pharmacia) for IgG tagged proteinsfollowed by SDS-PAGE analysis comparing to a known concentration ofprotein standard by Coomassie blue staining.

The first viral amplification supernatant was used to infect a spinnerculture (500 ml) of Sf9 cells grown in ESF-921 medium (ExpressionSystems LLC) at an approximate MOI of 0.1. Cells were incubated for 3days at 28° C. The supernatant was harvested and filtered. Batch bindingand SDS-PAGE analysis was repeated, as necessary, until expression ofthe spinner culture was confirmed.

The conditioned medium from the transfected cells (0.5 to 3 L) washarvested by centrifugation to remove the cells and filtered through0.22 micron filters. For the poly-His tagged constructs, the proteinconstruct were purified using a Ni-NTA column (Qiagen). Beforepurification, imidazole was added to the conditioned media to aconcentration of 5 mM. The conditioned media were pumped onto a 6 mlNi-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4° C. Afterloading, the column was washed with additional equilibration buffer andthe protein eluted with equilibration buffer containing 0.25 Mimidazole. The highly purified protein was subsequently desalted into astorage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at −80° C.

Immunoadhesin (Fc containing) constructs of proteins were purified fromthe conditioned media as follows. The conditioned media were pumped ontoa 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mMNa phosphate buffer, pH 6.8. After loading, the column was washedextensively with equilibration buffer before elution with 100 mM citricacid, pH 3.5. The eluted protein was immediately neutralized bycollecting 1 ml fractions into tubes containing 275 mL of 1 M Trisbuffer, pH 9. The highly purified protein was subsequently desalted intostorage buffer as described above for the poly-His tagged proteins. Thehomogeneity of the proteins was verified by SDS polyacrylamide gel (PEG)electrophoresis and N-terminal amino acid sequencing by Edmandegradation.

PRO301, PRO362 and PRO245 were also expressed in baculovirus infectedHigh-5 cells using an analogous procedure. High-5 cells were grown to aconfluency of 50% at 27° C., no CO₂, no penicillin and no streptomycin.For each 150 mm plate, 30 μg of pIE based vector containing PRO301,PRO362 or PRO245 was mixed with 1 ml Ex-Cell medium (Media: Ex-cell 401,1/100 L-Glu JRH Biosciences, #14401-78P, note: medium is lightsensitive), and in a separate tube, 100 μl of CELLFECTIN™ (GibcoBRL#10362-010) was mixed with 1 ml of Ec-Cell medium. The pIE1-1 and pIE1-2vectors are designed for constitutive expression of recombinant proteinsfrom the baculovirus ie1 promoter in stably-transformed insect cells(Cartier, J. L., et al., J. Virol. 68, 7728-7737)(1994). The plasmidsdiffer only in the orientation of the multiple cloning sites and containall promoter sequences known to be important for ie1-mediated geneexpression in uninfected insect cells as well as the hr5 enhancerelement. pIE1-1 and pIE1-2 include the ie1 translation initiation siteand can be used to produce fusion proteins.

The two solutions were combined and allowed to incubate at roomtemperature for 15 minutes. 8 ml of Ex-Cell media was added to the 2 mlof DNA/CELLFECTIN™ mix and is layered on High-5 cells previously washedwith Ex-Cell media. The plate was incubated in darkness for 1 hour atroom temperature. The DNA/CELLFECTIN™ mix was aspirated, and the cellswashed once with Ex-Cell to remove excess CELLFECTIN™. Fresh Ex-cellmedium (30 ml) was added and the cells incubated for 3 days at 28° C.The supernatant was harvested and the expression of PRO301, PRO362 orPRO245 was determined by batch binding in a manner similar to thatdescribed for Sf9 cells.

Example 17 Preparation of Antibodies that Bind PRO301, PRO362, PRO245and PRO1868

This example illustrates preparation of monoclonal antibodies which canspecifically bind PRO301, PRO362, PRO245 or PRO1868.

Techniques for producing the monoclonal antibodies are known in the artand are described, for instance, in Goding, supra. Immunogens that maybe employed include purified PRO301, PRO362, PRO245 and PRO1868, fusionproteins containing PRO301, PRO362, PRO245 and PRO1868, and cellsexpressing recombinant PRO301, PRO362, PRO245 and PRO1868 on the cellsurface. Selection of the immunogen can be made by the skilled artisanwithout undue experimentation.

Mice, such as BALB/c, are immunized with the PRO301, PRO362, PRO245 andPRO1868 immunogen emulsified in complete Freund's adjuvant and injectedsubcutaneously or intraperitoneally in an amount from 1-100 micrograms.Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (RibiImmunochemical Research, Hamilton, Mont.) and injected into the animal'shind foot pads. The immunized mice are then boosted 10 to 12 days laterwith additional immunogen emulsified in the selected adjuvant.Thereafter, for several weeks, the mice may also be boosted withadditional immunization injections. Serum samples may be periodicallyobtained from the mice by retro-orbital bleeding for testing in ELISAassays to detect PRO301, PRO362, PRO245 and PRO1868 antibodies.

After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of PRO301, PRO362, PRO245 and PRO1868. Three to four dayslater, the mice are sacrificed and the spleen cells are harvested. Thespleen cells are then fused (using 35% polyethylene glycol) to aselected murine myeloma cell line such as P3X63AgU.1, available fromATCC, No. CRL 1597. The fusions generate hybridoma cells which can thenbe plated in 96 well tissue culture plates containing HAT (hypoxanthine,aminopterin, and thymidine) medium to inhibit proliferation of non-fusedcells, myeloma hybrids, and spleen cell hybrids.

The hybridoma cells are screened in an ELISA for reactivity againstPRO301, PRO362, PRO245 or PRO1868. Determination of “positive” hybridomacells secreting the desired monoclonal antibodies against PRO301,PRO362, PRO245 or PRO1868 is within the skill in the art.

The positive hybridoma cells can be injected intraperitoneally intosyngenic BALB/c mice to produce ascites containing the anti-PRO301,anti-PRO362, anti-PRO245 or anti-PRO1868 monoclonal antibodies.Alternatively, the hybridoma cells can be grown in tissue culture flasksor roller bottles. Purification of the monoclonal antibodies produced inthe ascites can be accomplished using ammonium sulfate precipitation,followed by gel exclusion chromatography. Alternatively, affinitychromatography based upon binding of antibody to protein A or protein Gcan be employed.

Human PRO245 and PRO1868 cDNA were isolated from a human colonic cDNAlibrary by colony hybridization. Human IgG1 Fc fusion protein(immunoadhesins) of PRO245 (PRO245.Fc, also called JAM-IT.Fc or JAM2.Fc)and PRO1868 (PRO1868.Fc or JAM3.Fc) were prepared as described inAshkenazi et al. Curr. Opin. Immun. 9:195 (1997) and purified over aprotein A column (Amersham Pharmacia Biotech, N.J., USA). Identity wasverified by N-terminal sequence analysis.

BALB/c females were immunized and boosted with 10 μg of PRO245.Fc or8×His-tagged PRO1868 via footpad injection. Single clones were screenedagainst PRO245.Fc or 8×His-tagged PRO1868. Select clones were tested forcross reactivity against A33/JAM family members and human IgG Fc. Cloneswere titrated out to single cell densities and rescreened. Clone12D10.2F9 was discovered to be selectively reactive to JAM2 (PRO245) andnot JAM or JAM3. Clone MaJIR1 was found to be selectively reactive toJAM3 and not to JAM or JAM2. Both clones were isolated and used forascites generation. Abs were purified over a protein G column.

Anti-PRO245 antibody 12D10.2F9 was specific for interaction with PRO245expressing CHO cells and did not interact with human PRO301-expressingCHO cells (FIG. 58). Briefly, PRO245 cDNA was amplified by PCR from ahuman colon cDNA library (Clontech Laboratories, Palo Alto, Calif., USA)using primers specific for the 5′ and 3′ ends of the coding sequence.The fragment was purified and ligated into pSD5 expression vector,transfected into Chinese hamster ovary (CHO) cells and selected asdescribed in Lucas et al. Nuc. Acids Res. 24:1774 (1996). Stable cellclones were screened for antibody reactivity. As can be seen in FIG. 58,The anti-PRO245 antibody (12D10.2F9) did not bind to huJAM expressingCHO transfectant CuL8r. CuL8r does interact with the anti-huJAM antibody10A5.

Example 18 Isolation of cDNA Clones Encoding Human PRO1868 by ExpressionCloning

Identification of PRO1868 was done by transiently transfecting pooledcDNA libraries encoding secreted and transmembrane proteins into COScells grown on glass chamber slides. Twenty-four hours aftertransfection, PRO245 or PRO245-Fc fusions were added (0.5 μg/ml) andincubated for 30 minutes. PRO245/PRO245-Fc fusion binding was determined(Klein et al., Nature, 387:717 and 392:210 (1998)). Clones that werepositive for the ability to bind to PRO245/PRO245-Fc fusions wereselected for further characterization.

Example 19 Induction of Chondrocyte Re-Differentiation

The ability of the polypeptides of the invention to induceredifferentiation were tested in chondrocytes. Proteins with the abilityto induce redifferentiation of chondrocytes are useful for the treatmentof various bone and/or cartilage disorders such as, for example, sportsinjuries and arthritis.

Porcine chondrocytes were isolated by overnight collagenase digestion ofarticulary cartilage of metacarpophalangeal joints of 4-6 month oldfemale pigs. The isolated cells were then seeded at 25,000 cells/cm² inHam F-12 containing 10% FBS and 4 μg/ml gentamycin. The culture mediawas changed every third day and the cells were then seeded in 96 wellplates at 5,000 cells/well in 100 μl of the same media without serum and100 μl of the test PRO1868 polypeptide, 5 nM staurosporin (positivecontrol) or medium alone (negative control) was added to give a finalvolume of 200 μl/well. After 5 days of incubation at 37° C., a pictureof each well was taken and the differentiation state of the chondrocyteswas determined. A positive result in the assay resulted when theredifferentiation of the chondrocytes was determined to be more similarto the positive control than the negative control.

PRO1868 polypeptides tested positive for the ability to induceredifferentiation of chondrocytes.

Example 20 Overexpression of PRO1868 Polypeptides in Cancerous Tumors

In the present example, the expression level of PRO1868 polypeptides incancerous tissues was examined. Polypeptides that are overexpressed incancerous tumors may be useful as not only diagnostic markers for thepresence of one or more cancerous tumors, but also may serve astherapeutic targets for the treatment of those tumors.

For detection of overexpression of PRO1868 polypeptides, nucleic acidmicroarrays were used to identify differentially expressed genes indiseased tissues as compared to their normal counterparts. Using nucleicacid microarrays, test and control mRNA samples from test and controltissue samples are reverse transcribed and labeled to generate cDNAprobes. The cDNA probes are then hybridized to an array of nucleic acidsimmobilized on a solid support. The array is configured such that thesequence and position of each member of the array is known. For example,a selection of genes known to be expressed in certain disease states maybe arrayed on a solid support. Hybridization of a labeled probe with aparticular array member indicates that the sample from which the probewas derived expresses that gene. If the hybridization signal of a probefrom a test (disease tissue) sample is greater than hybridization signalof a probe from a control (normal tissue) sample, the gene or genesoverexpressed in the disease tissue are identified. The implication ofthis result is that an overexpressed protein in a diseased tissue isuseful not only as a diagnostic marker for the presence of the diseasecondition, but also as a therapeutic target for treatment of the diseasecondition. The methodology of hybridization of nucleic acids andmicroarray technology is well known in the art.

In the present example, cancerous tumors derived from various humantissues were studied for PRO1868 polypeptide-encoding gene expressionrelative to non-cancerous human tissue in an attempt to identify thosePRO1868 polypeptides which are overexpressed in cancerous tumors. Thespecific preparation of nucleic acids for hybridization and probes,slides, and hybridization conditions are all detailed in U.S.Provisional Patent Application Ser. No. 60/193,767, filed on Mar. 31,2000 and which is herein incorporated by reference. Two sets ofexperimental data were generated. In one set, cancerous human colontumor tissue and matched non-cancerous human colon tumor tissue from thesame patient (“matched colon control”) were obtained and analyzed forPRO1868 polypeptide expression using the above described microarraytechnology. In the second set of data, cancerous human tumor tissue froma variety of different human tumors, including lung and breast tumors,was obtained and compared to a “universal” epithelial control samplewhich was prepared by pooling non-cancerous human tissues of epithelialorigin, including liver, kidney, and lung. mRNA isolated from the pooledtissues represents a mixture of expressed gene products from thesedifferent tissues. Microarray hybridization experiments using the pooledcontrol samples generated a linear plot in a 2-color analysis. The slopeof the line generated in a 2-color analysis was then used to normalizethe ratios of (test:control detection) within each experiment. Thenormalized ratios from various experiments were then compared and usedto identify clustering of gene expression. Thus, the pooled “universalcontrol” sample not only allowed effective relative gene expressiondeterminations in a simple 2-sample comparison, it also allowedmulti-sample comparisons across several experiments.

Nucleic acid probes derived from the herein described PRO1868polypeptide-encoding nucleic acid sequences were used in the creation ofthe microarray and RNA from the tumor tissues listed above were used forthe hybridization thereto. A value based upon the normalizedratio:experimental ratio was designated as a “cutoff ratio”. Only valuesthat were above this cutoff ratio were determined to be significant.PRO1868 polypeptides of the present invention are significantlyoverexpressed in various human tumor tissues, for example lung andbreast tumors, as compared to a non-cancerous human tissue control.

These data indicate that the PRO polypeptides of the invention areuseful as both diagnostic markers and therapeutic targets for thetreatment of tumors.

Example 21 Induction of Cell Proliferation

A. Endothelial Cell Proliferation

The ability of polypeptides of the invention to induce proliferation inendothelial cells was tested in human umbilical vein endothelial cells(HUVEC, Cell Systems). Polypeptides with the ability to induceendothelial cell proliferation function as useful growth factors.

On day 0, pooled human umbilical vein endothelial cells (from celllines, maximum of 12-14 passages) were plated in 96-well plates at 1000cells/well per 100 microliter and incubated overnight in complete media[epithelial cell growth media (EGM, Clonetics), plus supplements: humanepithelial growth factor (hEGF), bovine brain extract (BBE),hydrocortisone, GA-1000, and fetal bovine serum (FBS, Clonetics)]. Onday 1, complete media was replaced by basal media [EGM plus 1% FBS] andaddition of PRO1868 polypeptides at 1%, 0.1% and 0.01%. On day 7, anassessment of cell proliferation was performed by ALAMAR BLUE™ assayfollowed by Crystal Violet. Results were expressed as a % of the cellgrowth observed with control buffer.

PRO1868 polypeptides tested positive in this assay for the ability toinduce proliferation of pooled human umbilical vein endothelial cells inculture, and as a result, to function as useful growth factors.

B. Human Coronary Artery Smooth Muscle Cell Proliferation

The ability of polypeptides of the invention to induce cellproliferation was tested in human coronary artery smooth muscle cells inculture. Polypeptides of the invention with the ability to induce cellproliferation are useful as growth factors.

On day 0, human coronary artery smooth muscle cells (from cell lines,maximum of 12-14 passages) were plated in 96-well plates at 1000cells/well per 100 microliter and incubated overnight in complete media[smooth muscle growth media (SmGM, Clonetics), plus supplements:insulin, human epithelial growth factor (hEGF), human fibroblast growthfactor (hFGF), GA-1000, and fetal bovine serum (FBS, Clonetics)]. On day1, complete media was replaced by basal media [SmGM plus 1% FBS] andaddition of PRO1868 polypeptides at 1%, 0.1% and 0.01%. On day 7, anassessment of cell proliferation was performed by ALAMAR BLUE™ assayfollowed by Crystal Violet. Results were expressed as a % of the cellgrowth observed with control buffer.

PRO1868 polypeptides tested positive in the assay for the ability toinduce proliferation of human coronary artery smooth muscle cells inculture and to function as a useful growth factor.

Example 22 PRO mRNA and Polypeptide Expression

A. In Situ Hybridization and Immunohistochemistry

Expression of PRO362, PRO245 and PRO1868 mRNA was evaluated by in situhybridization, immunohistochemistry and RT-PCR in various types oftissues.

For in situ hybridization, tissues were fixed (4% formalin),paraffin-embedded, sectioned (3-5 μm thick), deparaffinized,deproteinated (20 μg/ml) with proteinase K (15 minutes at 37° C.), andprocessed for in situ hybridization. Probes to the polypeptides of theinvention were produced by PCR. Primers included T7 or T3 RNA polymeraseinitiation sites to allow for in vitro transcription of sense orantisense probes from the amplified products. ³³P-UTP labeled sense andantisense probes were hybridized overnight (55° C.), washed (0.1×SSC for2 hours at 55° C.), dipped in NBT2 nuclear track emulsion (EastmanKodak, Rochester, N.Y.), exposed (4-6 weeks at 4° C.), and developed andcounterstained with hematoxylin and eosin. Representative paired brightand darkfield images are typically shown.

Immunohistochemical staining was performed on 5 mm thick frozen sectionsusing a DAKO Autostainer. Endogenous peroxidase activity was blockedwith Kirkegaard and Perry Blocking Solution (1:10, 4 minutes at 20 C.).10% NGS in TBS/0.05% Tween-20 (DAKO) was used for dilution and blocking.MAb 4F722.2 anti-STIgMA (anti-PRO362) or mouse IgG was used at 0.13mg/ml. Biotinylated goat anti-mouse IgG (Vector Labs), Burlingame,Calif.) was used at 1:200 and detected with Vector Labs Standard ABCElite Kit (Vector Labs, Burlingame, Calif.). Slides were developed usingPierce metal-enhanced diaminobenzidine (Pierce Chemicals, Rockford,Ill.). Dual immunohistochemistry for PRO362 (STIgMA) and CD68 expressionwas performed on frozen sections to demonstrate localization of STIgMAexpression to macrophages. mAb 4F7.22.2 anti-STIgMA and anti-CD68 mAbKP-1 from (DAKO) were utilized and detected by phycoerythrin and FITCmarkers, respectively.

1. Tissues Examined

Expression was examined in a wide variety of tissues and cell types fromhumans and other mammals.

a. Normal Tissue

Normal human adult tissues that were examined included tonsil, lymphnode, spleen, kidney, urinary bladder, lung, heart, aorta, coronaryartery, liver, gall bladder, prostate, stomach, small intestine, colon,pancrease, thyroid gland, skin, adrenal gland, placenta, uterus, ovary,testis, retina, and brain (cerebellum, brainstem, cerebral cortex).Normal human fetal tissues including E12-E16 week-old brain, spleen,bowel and thyroid were also tested. In addition, expression wasinvestigated in murine liver.

b. Inflamed Tissue

Inflamed tissues examined by in situ hybridization included tissues withchronic inflammatory disease such as lungs with chronic asthma, chronicbronchopneumonia, chronic bronchitis/chronic obstructive pulmonarydisease, kidneys with chronic lymphocytic interstitial nephritis, andlivers with chronic inflammation and cirrhosis due to chronic hepatitisC infection, autoimmune hepatitis or alcoholic cirrhosis.

c. Primary Neoplasms

Primary human neoplasms that were examined by in situ hybridization forPRO362, PRO245 and PRO1868 expression included breast carcinoma,pulmonary squamous cell carcinoma, pulmonary adenocarcinoma, prostaticadenocarcinoma, and colonic adenocarcinoma.

2. Results

a. PRO362 Expression

PRO362 was found to be expressed in mouse liver frozen sections (FIG.23), human liver frozen sections (FIG. 24) and a number of tissuemacrophage-like cells, including colon macrophages (FIG. 25A), Kupffercells (FIG. 25B), adrenal macrophages (FIG. 25C), Hofbauer cells (FIG.25D), synovial cells (FIG. 26), alveolar macrophages, residentmacrophages in the intestinal lamina propria and interstitialmacrophages in many tissues. PRO362 was also significantly expressed inbrain microglia. The expression of PRO362 was significantly increased inthese tissues when activated by the presence of neoplasia orinflammatory disease, including rheumatoid arthritis (FIG. 27),inflammatory bowel disease, chronic hepatitis (FIG. 28), pneumonia,chronic asthma (FIG. 29), glioma (FIG. 30) and bronchitis.

To further examine expression of PRO362, immunohistochemical stainingwas performed on various tissue types. Dual immunohistochemical stainingfor PRO362 and CD68 was performed on tissue macrophages, includingadrenal gland macrophages, liver Kupffer cells, brain microglial cells,and placental Hofbauer cells was performed to determine whether PRO362and CD68 are expressed in the same tissues.

PRO362 was found to be coexpressed with CD68 on adrenal glandmacrophages (FIG. 35), liver Kupffer cells (FIG. 36), brain microglialcells (FIG. 37), and placental Hofbauer cells (FIG. 38).

b. PRO245 Expression

PRO245 was found to be significantly localized to epithelial tissue andinflammed tissues.

(i) Normal Tissue

Expression of PRO245 mRNA in normal adult human tissues was significantin the high endothelial venules (HEVs) in tonsils and lymph nodes (FIG.31), the spermatogenic cells of the epithelium in the testicularseminiferous tubules (FIGS. 32I and J), and the intermediatetrophoblasts of the placenta.

Expression of PRO245 mRNA in normal human fetal tissues was significantin endothelial cells, but more specifically, was found in the vascularendothelium of small and large vessels (excluding capillaries), inmesenteric vessels, mural vessels of the bowel wall, and small vesselsof the developing mesenteric lymph nodes and thyroid.

Expression of PRO245 was not significant in the spleen, normal skin orforeskin, normal lung, thyroid, normal bowel, normal cardiac tissue oradrenal glands.

(ii) Inflamed Tissue

The expression of PRO245 was more extensive in tissues with chronicinflammatory diseases. In biopsies of lung with chronicbronchopneumonia, PRO245 mRNA was expressed in the endothelium of small-(FIGS. 32A and B), medium- (FIGS. 32C and D), and large-caliberarterioles (FIGS. 32E and F) present within or immediately adjacent tofoci of lymphocytic inflammation. PRO245 mRNA was not observed in normallung tissue (FIGS. 32G and H). Further, PRO245 was found to besignificantly expressed in the vascular endothelium in active or chronicinflammation in the following: arterioles, veins and capillaries fromtissues associated with chronic interstitial pneumonia, superficialdermal vessels of psoriatic skin from tissues associated with psoriasis,arterioles from tissues associated with chronic sclerosing nephritis,vascular endothelium and capillaries in inflammed foci from tissuesassociated with appendicitis, endothelium of numerous vessels, HEVs,capillaries, small arterioles and veins from tissues associated withtonsil and perifollicular sinuses, and capillaries in periarterialinterstitial tissue in aorta and aorta associated with atherosclerosis.PRO245 was not significantly expressed in aortic intima.

In biopsies of kidney with chronic lymphocytic interstitial nephritisand liver with chronic lymphocytic hepatitis, PRO245 expression wassignificant in the endothelium of arterioles in and adjacent to sites oflymphocytic inflammation. PRO245 expression was not significant inchronically inflammed or cirrhotic liver.

In biopsies of liver with chronic inflammation and cirrhosis, PRO245 wasnot significantly expressed.

PRO245 expression was not significantly expressed in inflammed largebowel or brain with meningitis.

(iii) Neoplastic Tissue

PRO245 expression was observed in the endothelium of small- andmedium-caliber arterioles in a number of primary neoplasms, includingcolonic adenocarcinoma, testicular carcinoma (FIGS. 33A and B),pulmonary adenocarcinoma (FIGS. 33C and D), mammary adenocarcinoma(FIGS. 33E and F) and significantly in prostatic adenocarcinoma andcolonic adenocarcinoma. PRO245 mRNA was found to be expressed in breastcarcinoma (FIG. 34). However, PRO245 was not significantly expressed inadjacent normal breast tissue, as shown in FIGS. 33G and H, where breastcarcinoma is denoted with an asterisk and normal breast tissue with anarrow. PRO245 expression is observed uniquely in vessels adjacent to thetumor (arrowheads), but not in normal tissue.

PRO245 expression was found in the vascular endothelium of epididymisand within areas of chronic lymphocytic inflammation in testicularcarcinomas or seminomas, in the vascular endothelium of tumor fociwithin areas of chronic lymphocytic inflammation of lung adenocarcinoma,in the vascular endothelium in tumor foci within areas of chroniclymphocytic inflammation in lung squamous cell carcinoma, in thevascular endothelium adjacent to tumor foci and within areas of chroniclymphocytic inflammation of breast carcinomas, and in areas adjacent tovascular endothelium in arterioles, veins, and capillaries inchondrosarcomas.

c. PRO1868 Expression

PRO1868 was found to be expressed on NK cells, CD8+ T cells anddendritic cells.

B. Reverse Transcription-Polymerase Chain (RT-PCR)

Reverse Transcription-polymerase chain reaction (RT-PCR) is a sensitivetechnique for mRNA detection and quantitation that consists of synthesisof cDNA from RNA by reverse transcription. To detect expression ofPRO1868, the presence of PRO1868 mRNA was detected by RT-PCR.

PRO1868 mRNA was significantly detected by reverse-transcriptasePCR(RT-PCR) in the T cell lines, J45 and Molt5, but not in the B celllines, JY, RPMI8866 and RAMOS (FIG. 39).

Example 22 Interaction of PRO245 with Specific Cell Types

As determined by flow cytometry, peripheral blood cells do notsignificantly express PRO245 (Table 6, top half). To determine whetherPRO245 interacted with discrete subsets of peripheral blood cells anumber of PRO245-cell assays were performed. These included magnetic orFACS sorting of PRO245-interacting cells. Peripheral blood was obtainedfor all experiments as described below.

A. Magnetic Sorting and Flow Cytometry

To determine whether PRO245 interacts with peripheral blood leukocytes,a biotinylated PRO245-human IgG fusion protein was generated, asdescribed below. PRO245-interacting peripheral blood leukocytes wereisolated using streptavidin-conjugated magnetic beads. Isolated cellswere then examined for surface CD-Ag expression. Results obtained usingbiotinylated PRO-245-human IgG fusion were compared to results usingbiotinylated human IgG.

Biotinylated PRO245-human IgG fusion proteins or human IgG1 proteinswere incubated for 1 hour at 4° C. with PBMC (10 μg/10⁷) in SerF buffer(10% FBS (v/v; Life Technologies) plus 0.1% NaN3 (w/v; Sigma-Aldrich,St. Louis, Mo.) in HBSS without phenol red or sodium bicarbonate (HBSS+;Life Technologies) buffered with 10 mM HEPES (Life Technologies), pH7.4). Cells were washed in SerF buffer and resuspended at 80 μl/10⁷cells. Streptavidin magnetic beads (Miltenyi Biotec) were added at 20μl/10⁷ cells and incubated for 15 minutes at 4 C, washed with SerFbuffer, resuspended at 500 ml/108 cells, and passed over a positiveselecting MACS column. Positively selected cells were eluted per themanufacturer's instructions, washed with SerF buffer, and analyzed byflow cytometry for surface CD Ags at 2×10⁵ cells per condition. The dataare presented as percentage positive, representing the percentage ofpositively stained cells in a total of 2×10⁵ cells collected perstaining condition for flow cytometry.

1. Protein Conjugation

PRO245-human IgG fusion, human IgG1, or PRO362-human IgG fusion werebiotinylated with 200 μg of EZ-Link sulfo-NHS-LC-biotin (Pierce) per 1mg of protein in PBS for 30 minutes at room temperature. Biotinylationwas quenched with the addition of (final concentration) 200 mM Tris, pH8, and incubated for 30 minutes at room temperature. Biotinylatedproteins were then dialyzed extensively against PBS and concentrated toa concentration of 2 mg/ml with Centricon-10 microconcentrators(Millipore, Bedford, Mass.).

Alexa-488 (Molecular Probes, Eugene, Oreg.) protein conjugation kit wasused per the manufacturer's instructions for the conjugation ofAlexa-488 onto PRO245-human IgG fusion or human IgG1.

2. Flow Cytometry

Cells for use in flow cytometric analysis were blocked for 30 minutes at4° C. with SerF buffer and stained with Abs to CD3, CD4, CD8, CD14, CD19or CD56, conjugated to either FITC, PE, or CyChrome (BD PharMingen, SanDiego, Calif.).

3. Results

The following four cell populations of peripheral blood leukocytes werefound to significantly interact with PRO245: T cells (CD3+), CD8+ cells,B cells (CD19+) and NK cells (CD56+). The percentage of cells that wereable to interact with PRO245 in a single experiment were as follows:20.99% for CD3+ cells, 6.68% for CD8+ cells, 9.66% for CD19+ cells, and36.89% for CD56+ cells. The percentage of cells that were able tointeract with the human IgG control were as follows: 2.39% for CD3+cells, 1.78% for CD8+ cells, 4.42% for CD19+ cells, and 6.69% for CD56+cells.

B. FACS Sorting and Flow Cytometry

PRO245-human IgG fusion protein-binding peripheral blood cells weresorted by FACS sorting.

For FACS sorting, cells were incubated (30 minutes at 4° C.) withAlexa-488-conjugated human IgG1 or PRO245-human IgG fusion protein (10μg/10⁶ cells) in a modified SerF buffer (SerF buffer with 5 μg/mlanti-CD16 Ab 3G8 (BD PharMingen) and 20 μg/ml human IgG1 (Calbiochem,San Diego, Calif.)), washed and sorted on an Elite ESP (Beckman Coulter,Miami, Fla.). In these conditions, Alexa-488-conjugated PRO245 or humanIgG was used as background. For competition assays, the competitor (20μg/10⁶ cells) was mixed with the cells for 20 minutes at roomtemperature in SerF buffer before Alexa-488-conjugated PRO245-human IgGfusion protein or human IgG were introduced. The cells were then washedand analyzed by flow cytometry as described above.

Of the cells that interact with PRO245-human IgG fusion protein(JAM-IT.Fc), 12.5% were CD3+ T cells, 32.4% were CD8+ T cells, and 50.4%were CD56+ NK cells. CD19+B cells were not detected in the FACS sortingassay. Of the CD56+ NK cells, 22.4% expressed CD3 and 40.2% expressedCD8. Of the CD8+ T cells, 23.5% expressed CD3 and 73.2% expressed CD56(FIG. 40).

TABLE 6 Expression of PRO245 on peripheral blood cells and binding ofperipheral blood cells to PRO245 Secondary Staining Primary Staining CD3CD4 CD8 CD14 CD19 CD56 PMN anti-PRO245 Positive 0.2% 0.1% 0.1% 0.7% 0.1% 0.3% 0.9% anti-PRO301 Positive 76.7% 73.9% 80.4%  99%  90%   85% 98.7% anti-mouse IgG Positive 0.25 0.1% 0.1% 0.7% 0.1%  0.3% 0.9% — — — — — —— — Percent of total PRO245 12.6% 1.1% 32.4% 0.3% 0.4% 50.4% NA bindingcells Percent of PRO245 binding 22.4% 0.2% 40.2% 0.5% 0.4% NA CD56positive cells Percent of PRO245 binding 23.5% 0.1% 0.6% 0.3% 73.2% NACD8 positive cells

C. Binding to Purified Cells

Purified B cells, neutrophils, CD14+ monocytes, peripheral blooddendritic cells (PBDCs) from Clonetics (San Diego, Calif.), peripheralblood CD56+ NK cells obtained by negative selection, and J45, a CD3+ Tcell line, were analyzed for their ability to interact withAlexa-488-conjugated PRO245-human IgG fusion protein by flow cytometry.The ability to interact with Alexa-488-conjugated human IgG1 protein wasanalyzed at the same time as a control.

Blood was obtained from healthy adult volunteers by venous puncture andseparated using Ficoll-Plaque PLUS (Amersham Pharmacia Biotech) per themanufacturer's instructions. PBMC were obtained from the interface,washed in cold PBS, lysed (with 0.2% NaCl for 30 seconds and neutralizedwith 1.6% NaCl) as needed, counted, and kept on ice at 5×10⁷ cells/mluntil use. By flow cytometric analysis, no contaminating platelets wereobserved in the purified PBMC fractions. Neutrophils were obtained fromthe pellet after lysis of contaminating RBCs. Neutrophils were washed incold PBS, counted, and kept at 5×10⁷ cells/ml until use on ice. Toisolated peripheral blood subsets, “untouched” MACS kits (MiltenyiBiotec, Auburn, Calif.) were used following the manufacturer'sinstructions.

Purified B cells, neutrophils and CD14+ monocytes did not interact withPRO245-human IgG fusion protein as detected by flow cytometry. However,a number of other cell types were found to interact with PRO245.Fc. FIG.41 shows that PRO245.Fc interacts with CD56+ NK cells. This interactionwas specific, as it was blocked by the addition of an anti-PRO245antibody. PRO1868 (also called 77624, JAM3 and SHATr) was found to blockthe interaction of PRO245 and CD56+ NK cells (FIG. 46, bottom, and FIG.53, lower right). Addition of unlabeled, His tagged PRO1868 (JAM3)blocked the shift in fluorescence observed with the addition ofPRO245.Fc. On the other hand, as can be seen in FIG. 53, upper right,addition of PRO301 does not block the interaction of PRO245 and NKcells.

Peripheral blood dendritic cells (PBDCs) also interact with, but do notexpress PRO24 (FIG. 42). PBDC were obtained from Clonetics. FIG. 41Ishows that PRO245.Fc (solid line) interacts strongly with the PBDCscompared to human IgG1 (shaded histogram). However, PBDS were notobserved to express PRO245 (FIG. 41II; mouse IgG-filled histogram;anti-PRO245 antibody 12D10.2F9-solid line).

In addition, J45 T cells, which have no detectable surface expression ofPRO245, were found to interact with PRO245 (FIGS. 43 and 44).Interaction between Alexa-488 conjugated PRO245-Fc fusion protein andJ45 cells was detected by a shift in the peak of fluorescence whencompared to the conjugated human IgG1 (FIG. 44). The shift was blockedby addition of anti-PRO245 antibody (FIG. 44). The interaction betweenAlexa-488 conjugated PRO245-Fc fusion protein and J45 cells was alsoinhibited by unlabeled PRO1868 (His-PRO1868 protein) (FIG. 45). Further,an anti-PRO1868 antibody (MaJIR1) was found to inhibit PRO245 dependentJ45 adhesion, while mouse IgG had no effect on adhesion (FIG. 52).

Accordingly, PRO245-interacting cell types were as follows: CD56+ cells,including CD56+ NK cells, CD56+CD3+ NK/T cells, CD56+CD3+ CD8+ cytolyticT cells, PBDCs and J45 T cells. Further, excess PRO1868 proteininhibited PRO245 binding to J45 and CD56+ NK cells, and anti-PRO1868antibodies inhibited PRO245 binding to CD56+ NK cells.

D. Plate-Based Adhesion Assay

For plate-based analysis of cells that are able to interact with PRO245,microtitre wells (NUNC Maxisor 96-well plates; VWR, Scientific Products,Brisbane, Calif.) were coated with conditions at 50 μl/well (in HBSS+),10 μl/ml for 2 hours at room temperature, unless otherwise noted. Foradhesion assays, 50 μl of 10 μg/ml goat anti-human IgG1 Fc-specific Ab,for example PRO245-human IgG fusion protein, was first coated andblocked before the addition of conditions in binding/blocking buffer(BBB; HBSS+containing 10% (v/v) FBS) for 1 hour at room temperaturebefore the addition of coating condition. Cells (5×10⁶ cells/ml in BBB)were treated (10 minutes at 37° C. with 5% CO₂) with 5 mg/ml2′,7′-bis-(2-carboxyethyl)-5 (and -6)-carboxyfluorescein, acetyoxymethylesther (BCECF AM) (Molecular Probes), washed, and allowed to adhere tocoated wells (2×10⁵ cells/well in BBB) for 1 hour at 37° C./5% CO₂.

Plates were read on a SpectraMax fluorescence plate reader (MolecularDevices, Sunnyvale, Calif.) for total applied fluorescence, gentlywashed three times (by aspiration with a 28-gauge needle), and read fortotal adherent fluorescence. Percentage of adherence was calculatedusing the following equation: ((total fluorescence of adherent)/(totalfluorescence of applied))×100. Blank wells consisted of BBB-coated wellsexposed to BCECF AM-labeled J45 cells. Values obtained from the blankwells (percentage of adherence) were subtracted from all experimentalconditions to derive a final value.

Using the plate-based adhesion assay, J45 T cells were found to adhereto PRO245-human IgG fusion (VJ2.Fc) coated wells (FIG. 47). Anti-PRO245antibodies, but not mouse IgG, inhibited adhesion of J45 cells toPRO245-human IgG1 fusion protein, indicating that the interaction isspecific (FIG. 47).

Example 23 Identification of Receptors for PRO245

To identify the protein in PRO245-interacting cells that is responsiblefor the interaction, immunoprecipitation studies were performed.

A. CD56+ NK Cells and J45 Cells

To isolate the cell surface receptor on J45 or NK cells for PRO245,PRO245 interacting cells were biotinylated and then lysed. Thesupernatants from the lysed cells were subjected to immunoprecipitationwith a Fc-cross-linked PRO245-human IgG fusion protein A matrix. Theprecipitates were analyzed by Western blotting.

1. Biotinylation

For biotinylated conditions, cells were first washed in HBSS+ beforebeing biotinylated (200 μg/10⁶ cells) with sulfo-NHS-LC-biotin for 30minutes at 4° C. Cells were washed with TBS for 30 minutes at 4° C. toquench the biotinylation.

2. Lysis

Cells were lysed (108 cells/ml) with lysis buffer (HBSS+ containing 1%Triton X-100 and 1 Complete-Mini EDTA free protease inhibitor tablet(Roche Biochemicals, Indianapolis, Ind.) per 7 ml of lysis buffer) for30 minutes at 4° C. Lysates were spun at 22,000×g for 1 hour at 4° C.and 0.2 μm filtered. Lysates were precleared for 2 hours at 4° C. with 5μl/10⁶ cells of recombinant protein A beads (Amersham PharmaciaBiotech).

3. Immunoprecipitation

Cleared lysates were 0.2 μm filtered and incubated for 2 hours at 4° C.with 5 μg/10⁶ cells of either PRO245-human IgG fusion protein or humanIgG1, conjugated to protein A matrix using the ImmunoPure Protein A IgGPlus Orientation kit (Pierce). Beads were pelleted and washed with lysisbuffer and denatured by the addition of 15 μl/10⁶ cells of nonreducingSDS sample buffer (Standard sample buffer with 2 mM iodoacetamide, butwithout DTT or 2-mercaptoethanol) and boiled for 3 minutes at 100° C.

4. Western Blotting

Samples at a concentration of 15 μl/lane were resolved on a 4-20%Bio-Rad Tris-HCl Ready Gel (Bio-Rad, Hercules, Calif.) and transferredonto 0.2-μm Protran nitrocellulose membrane (Schleicher & Schuell,Keene, N. H.) at 100 mA for 2 hours at 4° C. Blots were blocked for 1hour in Blotto (TBS containing 5% nonfat milk and 0.05% TWEEN™ 20;Bio-Rad). For biotinylated samples, HRP-conjugated streptavidin (Pierce)was used at 0.5 μg/ml for 30 minutes at room temperature. Fornonbiotinylated samples, anti-PRO1868 antibodies (MaJIR1) was used at 10μg/ml in Blotto and incubated for 1 hour at 25° C. before theapplication of 1 μg/ml HRP-conjugated goat anti-mouse IgG (CaltagLaboratories, Burlingame, Calif.) in Blotto for 30 minutes at roomtemperature. Blots were washed thoroughly with TTBS (TBS containing0.05% Tween 20 (and developed with the ECL Plus reagent (AmershamPharmacia Biotech) before exposing onto Kodak BioMax ML film anddevelopment with Kodak M35A X-OMAT Film Processor (Eastman Kodak).

5. Results

Immunoprecipitation of biotinylated samples with an Fc-cross-linkedPRO245-human IgG fusion protein A matrix and analysis by Westernblotting allowed identification of a single streptavidin-reactive bandof about 40 kDa that interacts with PRO245-human IgG fusion (FIG. 48).The 40 kDa band was not present in immunoprecipitations performed withan Fc-cross-linked human IgG protein A matrix, nor inPRO245-immunoprecipitations performed with an Fc-cross-linked human IgGprotein A matrix in the non-PRO245-binding Ramos/HH B cell line.

To determine whether the 40 kDA band represented PRO1868,immunoprecipitation was performed on non-biotinylated samples fromRamos/HH cells (non-PRO245 interacting), MOLT4 cells (PRO245 binding)and PBMCs (PRO245 binding) with PRO245-human IgG fusion protein Amatrixes. The precipitates were analyzed by immunoblotting withanti-PRO1868 antibodies. The immunoblotting verified that the 40 kDaPRO245-interacting band represented PRO1868 (FIG. 49).

6. Confirmation of Binding Between PRO245 and PRO1868 by ELISA

Using anti-PRO1868 antibodies, purified PRO245 and PRO1868 fusionproteins, an interaction between PRO245 and PRO1868 was confirmed in aplate-based assay. Plate-bound PRO1868-Fc fusion protein (JAM3.Fc) or acontrol human PRO301-Fc fusion protein (huJAM.Fc) were exposed tobiotinylated PRO245-Fc fusion protein in the presence of 0.25 μg/wellmouse IgG or an anti-PRO1868 Ab (FIG. 50). Streptavidin HRP was used todetect binding between the PRO1868-Fc fusion protein-coated wells toPRO245-Fc biotin. Alternatively, PRO245-Fc fusion was captured onto aplate, and biotinylated PRO1868-Fc fusion was used at specificconcentrations to examine the PRO245-PRO1868 interaction (FIG. 51).Further, inhibition of such a plate-based interaction between PRO245 andPRO1868 by anti-PRO1868 antibodies and anti-PRO245 antibodies wastested.

For ELISA, the plates were blocked after condition coating with BBB for30 minutes at room temperature and incubated with binding conditions for1 hour at room temperature. For conditions requiring EDTA, a modifiedBBB (HBSS without calcium and magnesium containing EDTA instead of thenormal HBSS+) was used throughout the experiment. Plates were washedthree times, incubated with 1 μg/ml streptavidin HRP (Pierce) for 30minutes at room temperature, and assessed via color development usingthe tetramethylbenzidine substrate (Kirkegaard & Perry Laboratories,Gaithersburg, Md.) and read on the ThermoMax Microplate Reader(Molecular Devices).

An interaction between PRO245 and PRO1868 was identified through theplate-based assays (FIGS. 50 and 51). FIG. 50 shows that PRO1868.Fc(JAM3.Fc) coated wells demonstrated PRO245.Fc binding while PRO301.Fccoated wells did not. Mouse IgG (MIgG) had no effect on binding whilethe anti-PRO1868 antibody (MaJIR1) inhibited PRO245 binding. WhenPRO245.Fc was bound to the plate (FIG. 51), interaction of PRO245.Fc andPRO1868.Fc was again observed. In addition, the anti-PRO1868 antibodyMaJIR1 was again able to inhibit the interaction, while the mouse IgGhad no effect.

B. Panel of Potential Receptors

PRO245 polypeptide was incubated with a panel of potential receptormolecules for the purpose of identifying the receptor/ligandinteraction. The identification of a ligand for a known receptor, areceptor for a known ligand or a novel receptor/ligand pair is usefulfor a variety of indications including, for example, targeting bioactivemolecules (linked to the ligand or receptor) to a cell known to expressthe receptor or ligand, use of the receptor or ligand as a reagent todetect the presence of the ligand or receptor in a composition suspectedof containing the same, wherein the composition may comprise cellssuspected of expressing the ligand or receptor, modulating the growth ofor another biological or immunological activity of a cell known toexpress or respond to the receptor or ligand, modulating the immuneresponse of cells or toward cells that express the receptor or ligand,allowing the preparation of agonists, antagonists and/or antibodiesdirected against the receptor or ligand which will modulate the growthof or a biological or immunological activity of a cell expressing thereceptor or ligand, and various other indications which will be readilyapparent to the ordinarily skilled artisan.

A PRO245 polypeptide of the present invention suspected of being aligand for a receptor is expressed as a fusion protein containing the Fcdomain of human IgG (an immunoadhesin). Receptor-ligand binding isdetected by allowing interaction of the PRO245 immunoadhesin polypeptidewith cells (e.g. COS cells) expressing candidate receptors, includingthe PRO1868 polypeptide receptor, and visualization of boundimmunoadhesin with fluorescent reagents directed toward the Fc fusiondomain and examination by microscope. Cells expressing candidatereceptors are produced by transient transfection, in parallel, ofdefined subsets of a library of cDNA expression vectors, for example,encoding PRO1868 polypeptides, that may function as receptor molecules.Cells are then incubated for 1 hour in the presence of the PRO245polypeptide immunoadhesin being tested for possible receptor binding.The cells are then washed and fixed with paraformaldehyde. The cells arethen incubated with fluorescent conjugated antibody directed against theFc portion of the PRO245 polypeptide immunoadhesin (e.g. FITC conjugatedgoat anti-human-Fc antibody). The cells are then washed again andexamined by microscope. A positive interaction is judged by the presenceof fluorescent labeling of cells transfected with cDNA encoding aparticular PRO1868 polypeptide receptor or pool of receptors and anabsence of similar fluorescent labeling of similarly prepared cells thathave been transfected with other cDNA or pools of cDNA. If a definedpool of cDNA expression vectors is judged to be positive for interactionwith a PRO245 polypeptide immunoadhesin, the individual cDNA speciesthat comprise the pool are tested individually (the pool is “brokendown”) to determine the specific cDNA that encodes a receptor able tointeract with the PRO245 polypeptide immunoadhesin.

In another embodiment of this assay, an epitope-tagged potential ligandPRO245 polypeptide (e.g. 8 histidine “His” tag) is allowed to interactwith a panel of potential receptor polypeptide molecules that have beenexpressed as fusions with the Fc domain of human IgG (immunoadhesins).Following a 1 hour co-incubation with the epitope tagged PRO245polypeptide, the candidate receptors are each immunoprecipitated withprotein A beads and the beads are washed. Potential ligand interactionis determined by western blot analysis of the immunoprecipitatedcomplexes with antibody directed towards the epitope tag. An interactionis judged to occur if a band of the anticipated molecular weight of theepitope tagged protein is observed in the western blot analysis with acandidate receptor, but is not observed to occur with the other membersof the panel of potential receptors.

Using these assays, the following receptor/ligand interactions have beenherein identified: PRO245 (DNA35638-1141) binds to PRO1868(DNA77624-2515).

C. JAM Family Proteins

Flow cytometry analysis was performed to further investigate theinteractions of members of the JAM protein family. PRO245 was expressedin CHO cells as described in Example 14. The PRO245-expressing CHO cellswere then incubated with His-tagged JAM proteins, including PRO245,PRO301, and PRO1868. Binding of His-tagged PRO362, PRO1868 or PRO301proteins to PRO245-expressing CHO cells were analyzed by flow cytometry.

For binding of PRO1868 to PRO245-expressing CHO cells, 5 μg/ml ofPRO1868-HIS (SHATr.His) tagged protein was incubated withPRO245-expressing CHO cells. PRO1868 (SHATr.His) was able to interactwith PRO245-expressing CHO cells (FIG. 54). Varying competitor proteinswere examined for their ability to inhibit binding of PRO1868 to PRO245.PRO1868 protein (SHATr.His) and anti-PRO245 antibody (12D10.2F9) wereable to compete with PRO1868-HIS tagged protein for binding of PRO245 onthe surface of CHO cells (FIG. 54). In contrast, PRO301.Fc, PRO362.Fc,mouse IgG and His control were not able to inhibit binding (FIG. 54).

Based on the results described above, PRO245 interacts with PRO1868.

Example 24 Involvement of STIGMA (PRO362) in Chronic Inflammation

The novel macrophage associated receptor with homology to A33 antigenand JAM1 was cloned as described in Example 2 and below, and wasidentified as a single transmembrane Ig superfamily member macrophageassociated (STIgMA or PRO362 or JAM4).

STIgMA is expressed as two spliced variants, one containing anN-terminal IgV like domain and a C-terminal IgC2 like domain and aspliced form lacking the C-terminal domain. Both receptors have a singletransmembrane domain and a cytoplasmic domain containing tyrosineresidues which are constitutively phosphorylated in macrophages invitro.

The present study demonstrates that STIgMA is selectively expressed on asubset of tissue resident macrophages, and is associated with chronicinflammation.

Materials and Methods

Cells

Blood was obtained from healthy adult volunteers by venous puncture andseparated using Ficoll-Paque PLUS (Amersham Pharmacia Biotech) permanufacturers instruction. PBMCs were obtained from the interface,washed in cold PBS, lysed with 0.2% NaCl for 30 s and neutralized with1.6% NaCl. Cells were counted and kept on ice until use. To isolateperipheral blood subsets, untouched MACS kits (Miltenyi Biotech, Auburn,Calif.) were used following the manufacturers instructions. To culturedifferentiated macrophages, negatively selected monocytes weretransferred to 6 well culture dishes in HGDMEM containing 20% fetalbovine serum and 10% human serum. Medium was replaced at day 5. For flowcytometric analysis, cells were dissociated from the culture dish usingice-cold cell dissociation solution (Sigma). Lysates for Western blotanalysis were prepared by adding 0.5 ml lysis buffer directly to thewells. Lysates were mixed with sample buffer containing SDS andbeta-mercaptoethanol, run on a Tris-Glycine gel and transferred to anitrocellulose membrane.

Flow Cytometry

Cells for use in flow cytometric analysis were blocked for 30 min at 4 Cwith PBS containing 2% fetal bovine serum and 5 μg/ml human IgG(Calbiochem, San Diego, Calif.). Nex, cells were incubated with 3C9, ananti-STIgMA (anti-PRO362) monoclonal antibody. After washing in PBS,cells were stained with phycoerythrin (PE)-conjugated antibodies toCD11b, CD 14, CD163, CD15, CD68 were obtained from Pharmingen.

Cell-Cell Adhesion Studies

A pRK expression vector containing full length STIgMA was stablyexpressed in a human Jurkat T-cell line using neomycin selection andautoclone sorting as described elsewhere. Cells were preloaded with thefluorescent dye BCECF (Molecular Probes, Oregon) and added to a 96 wellMaxisorb plate (CORNING™) coated with a monolayer of human umbilicalvein endothelial cells (HUVEC) treated with or without 10 ng/mlTNFalpha. Cells were gently washed by loading the wells with incubationbuffer (HBSS contained 10 mM CaCl, 10 mM magnesium and 1.5 mM NaCl)followed by inverting the plate on a piece of blotting paper. After 3washes, fluorescence was counted in a fluorospectrometer. Thefluorescent readout is representative of the number of cells that remainadherent to the HUVEC cells.

Northern Blot Analysis

Multiple tissue Northern blots (CLONTECH) were probed with a ³²P labeledprobe of random-primed full-length STIgMA cDNA using Ambion kitaccording to manufacturers recommendations. Blots were exposed to aphosphorimaging screen and analyzed with a Storm phosphorimager.

Real Time RtPCR Analysis

For quantitative PCR analysis (TAQMAN™), total mRNA from human tissuesor primary cells (100 ng) was recommended (PerkinElmer Life Sciences)with primers based on the coding sequence of STIgMA.

Fc- and His-Fusion Protein Production

Human STIgMA was cloned into the baculovirus expression vector pHIF(Pharmingen). The HIS-tagged STIgMA fusion protein consisted of theextracellular domain of STIgMA fused to 8 histidines. His-tagged fusionprotein was purified from the supernatant of baculovirus-infected insectcells grown in suspension using nickel affinity resin.

Monoclonal and Polyclonal Antibody Production

BALBc females were immunized and boosted with 10 μg STIgMA-His8 viafootpad injections, as previously described. Single clones were screenedagainst STIgMA (PRO362)-His by ELISA. Selected clones selected cloneswere tested against JAM family members and human IgG Fc. Clones weretitrated out to single cell densities and rescreened. Clone 3C9 (IgG1)was found to be selectively reactive to STIgMA. Clones were used forascites generation and purified over protein G (Amersham PharmaciaBiotech); protein concentration was determined using the Pierce BCAreagent (Pierce, Rockford, Ill.).

Polyclonal antibodies were generated by injecting 150 μg STIgMA-His inNew Zealand Rabbits. Serum titers were determined by ELISA. Serum wascollected at the peak of circulating IgG levels and purified over aprotein A column.

In Situ Hybridization

PCR primers (upper 5′-TCTCTGTCTCCAAGCCCACAG (SEQ ID NO: 35), and lower,5′-CTTTGAGGAGTCTTTGACC (SEQ ID NO: 36)) were designed to amplify a 700bp fragment of huJAM4. Primers included T7 or T3 RNA polymeraseinitiation sites to allow for in vitro transcription of sense orantisense probes, respectively, from the amplified products. Normalhuman tissues included tonsil, lymph node, spleen, kidney, lung andheart. Tissues with chronic inflammatory disease included lung withchronic asthma, chronic bronchitis, livers with chronic inflammation andcirrhosis due to chronic hepatitis C infection. Tissues were fixed in 4%formalin, paraffin embedded, sectioned (3-5 μm thick) deparaffinized,deproteinated with 20 μg/ml proteinase K (15 min at 37° C.) andprocessed for in situ hybridization as described elsewhere.

Immunohistochemistry

Immunohistochemical staining was performed on 5-μm thick frozen sectionsusing a DAKO autostainer. Endogenous peroxidase activity was blockedwith Kirkegaard and Perry blocking solution (1:10, 4 min 20° C.). Normalgoat serum (NGS) at 10% in TBS/0.05% Tween-20 was used for dilution andblocking. Mab 3C9 was used at 1 ug/ml. Slides were developed usingmetal-enhanced diaminobenzidine (Pierce Chemicals). Forimmunofluorescence staining of sections, sections were blocked withPBS/10% NGS and incubated with mAb 3C9 for 1 hr at 20° C. A rabbit-antimouse FITC-labeled secondary antibody conjugated to FITS was used asdetections agent. For double staining procedure, sections weresubsequently stained with a PE-conjugated monoclonal antibody to humanCD68.

Results

Molecular Cloning of Human STIgMA

HuSTIgMA was cloned from a human fetal cDNA library using degenerateprimers recognizing conserved Ig domains of human JAM1. Sequencing ofseveral clones revealed an open reading frame of 400 amino acids. Blastsearches confirmed similarity to Z39Ig, a type 1 transmembrane protein(Langnaese et al., Biochim Biophys Acta 1492 (2000) 522-525. Theextracellular region of STIgMA consisted of 21 g-like domains,comprising an N-terminal V-set domain and a C-terminal C2-set domain.Using 3′ and 5′ primers, a splice variant of STIgMA, STIgMA short whichlacks the membrane proximal IgC domain and is 50 amino acids shorter wascloned.

Cloning of Murine STIgMA and Sequence Comparison with Human STIgMA

The murine expressed sequence tags (EST) database was searched using thefull open reading frame of huSTIgMA (PRO362) and the tblastn algorithm.DNA sequencing of 3 clones gave rise to identical complete open readingframes of 280 amino acids. Primers to the 3 prime regions were used toclone a full length transcript from a mouse spleen library. The murineclone resembled the spliced form of hu STIgMA in that, it lacked theC-terminal Ig-like domain. The extracellular IgV-domain was wellconserved between the human and murine receptor with 93% identity. Themurine cytoplasmic domain was poorly conserved being 20 amino acidsshorter than its human counterpart and was 40% identical.

STIgMA is Expressed on a Subset of Resident Macrophages in DiverseTissues and its Expression is Increased in Inflammation

Northern blot analysis of huSTIgMA showed two transcripts of 1.8 and 2.2kb (FIG. 57) with highest expression in the adrenal gland, lung andplacenta, and lower expression in heart, spinal chord, thyroid gland,mammary gland and lymph node. In all tissues, the 2.2 kb transcript wasthe most abundantly expressed transcript and presumably, encodes thelong form of STIgMA.

TAQMAN™ Real-Time PCR Analysis

To identify specific cell lines expressing STIgMA, real-timequantitative PCR and primers/probes specific for the N-terminal Igdomain was used. Low but detectable mRNA expression was found in themyeloid cell line HL-60 treated with PMA and the monocytic cell lineTHP-1. Expression was absent in B- and T-cell lines (FIG. 58A).

STIgMA (PRO362) Expression on Differentiated Monocytes.

In order to establish details of when STIgMA was expressed indifferentiating monocytes/macrophages, we determined STIgMA mRNA levelsin non-adherent monocytes and in adherent monocytes, induced todifferentiate in the presence of human autologous serum. STIgMA mRNAlevels gradually increased over time and reached maximum levels at 7days following plating (FIG. 58B). At this differentiation stage, mRNAlevels were 100 fold higher as compared to those in undifferentiatedmonocytes.

Western blotting of monocyte/macrophage lysates showed an increase inSTIgMA protein expression (FIG. 58C) in parallel with the increase inSTIgMA mRNA expression, indicating that STIgMA was expressed whenmonocytes differentiated to form macrophages. A band of 48 kDa and aband of 40 kDa appeared on the blot, presumably representing the longand the short forms of human STIgMA.

Molecular Characterization of STIgMA (PRO362)

STIgMA migrated similarly under reduced and non-reduced conditionsindicating that it was expressed as a monomer (FIG. 59A). Only slightchanges in migration patterns were observed when STIgMA wasdeglycosylated using PNGase F, indicating insignificant N-glycosylation.STIgMA was phosphorylated when STIgMA overexpressing cells were treatedwith pervanadate (FIG. 59B). Phosphorylated STIgMA migrated as aslightly higher Mw protein (55 kDa). In human HEK 293 cells,tyrosine-phosphorylated STIgMA cytoplasmic domain does not recruit Sykkinase (results not shown).

Flow Cytometry Analysis of STIgMA Expression on Peripheral BloodMononuclear Cells

In order to determine the expression pattern of STIgMA in circulatingleukocytes, flow cytometric analysis was performed on lymphocytesisolated from blood from a healthy donor using monoclonal anti-humanSTIgMA antibody 3C9 directly conjugated with ALEXA™ A488.Counterstaining was performed with PE conjugate antibodies to severalimmune-cell surface antigens. STIgMA was absent on the surface of allleukocytes, including B-T-Nk cells, monocytes and granulocytes (FIG.60). STIgMA was however expressed on monocytes cultured for 7 days inmacrophage differentiation medium.

Regulation of STIgMA Expression in Monocytes

In order to study the regulation of expression of STIgMA, 7 daymacrophages were cultured in the presence of various pro- andanti-inflammatory cytokines and STIgMA expression levels were determinedby real-time PCR or flow analysis. Expression of STIgMA mRNA wasincreased after treatment of macrophages for 2 days with IL-10 and TGF-βand down regulated by IL-4, IL13 and LPS (FIG. 61A). Treatment withdexamethasone increased expression to 5 fold compared to controlnon-treated macrophages. In order to determine the regulation ofcell-surface expressed STIgMA, flow cytometry was performed onperipheral blood monocytes treated with various cytokines anddexamethasone for 5 days. STIgMA was detected using monoclonal antibodyclone 3C9 conjugated to ALEXA™ A488. Cells were co-stained with antiCD-14 antibodies. Increased surface expression of STIgMA was foundfollowing treatment of monocytes with IL-10 and LPS for 5 days (FIG.61B). A dramatic increase in surface STIgMA expression was found aftertreatment with dexamethasone.

Subcellular Distribution of STIgMA

In order to study the subcellular distribution of STIgMA, MDMs were keptin culture for 15 days after which they were fixed and stained with amonoclonal antibody (clone 3C9) or polyclonal rabbit antibody 4F7followed by FITC conjugated secondary antibody and a PE-labeled antiCD63 antibody. Confocal microscopy showed high expression of STIgMA inthe perinuclear cytoplasm, overlapping with the expression of thelysosomal membrane protein CD63 (FIGS. 62A,B). STIgMA was also expressedin the leading and trailing edges of the macrophages where its stainingpattern did not overlap with that of CD63.

Expression of STIgMA in Normal and Disease Tissues

STIgMA expression in tissue resident macrophages and changes in itsexpression in tissues with chronic inflammatory diseases was studied.Using in situ hybridization, STIgMA mRNA expression was determined onpanels of paraformaldehyde-fixed human tissues. High expression levelswere found in alveolar macrophages obtained from a lung autopsy of apatient with pneumonia or chronic asthma (FIG. 63A-D). High mRNAexpression was found in Kupffer cells in the liver of a patient withchronic hepatitis (FIGS. 63E, F).

In a previous study (Walker, Biochimica et Biophysica Acta 1574 (2002)387-390), and in electronic screening of libraries, high expression ofSTIgMA mRNA was found in the synovium of patients with rheumatoidarthritis. Therefore, the expression pattern of STIgMA in synoviumobtained from patients with rheumatoid arthritis, osteoarthritis anddegenerative bone disease was studied. High expression of STIgMA mRNAwas found in synovial cells obtained from a patient with osteoarthritis(FIG. 64A-D). Synovial cells in the superficial layers had the highestexpression of STIgMA (FIG. 64D). In addition, polyclonal antibody 6F1was used to study STIgMA expression in frozen sections of human synoviumobtained from a patient with rheumatoid arthritis. STIgMA was expressedin a subset of synovial cells (20-40%) and in tissue macrophages in thesynovium (FIG. 65A-C). These cells were, most likely, type Amacrophage-like synovial cells. Staining was absent in control synovium(FIG. 65D).

Expression of STIgMA protein was found on macrophages in a number ofdifferent tissues. Frozen sections prepared from CHO cells stablyexpressing STIgMA show membrane localization of STIgMA (FIG. 66A).STIgMA protein was found in alveolar macrophages (FIG. 66B), histiocytesin the lamina propria of the small intestine (FIG. 66C), Hofbauer cellsin the placenta (FIG. 66D), macrophagesin the adrenal gland (FIG. 66E)and Kupffer cells in the liver (FIG. 66F).

Atherosclerotic plaques contained a high number of macrophages ormacrophage-foam cells that adhered tightly to the luminal wall of theaorta. Considering a role for STIgMA in macrophage-endothelium adhesion,the expression of STIgMA in atherosclerotic plaques was studied.Alternate sections of plaques were stained with anti CD63 (FIGS. 67A andB) or anti-STIgMA (FIGS. 67C and D). Overlapping staining patterns ofanti-CD63 and STIgMA was found on foam cells aligning the vessel wallindicating a role for STIgMA in atherosclerosis.

In order to determine whether STIgMA was selectively expressed onmacrophages, double staining immunofluorescence was performed on heartinterstitial macrophages (FIG. 68). As shown in the overlay (FIG. 68C)most of the interstitial macrophages positive for STIgMA were alsopositive for CD68. Not all CD68 positive macrophages were positive forSTIgMA, indicating that the latter was specific for a subtype of tissueresident macrophages.

In order to quantitatively determine mRNA expression levels ininflammatory bowel disease (IBD) syndrome, mRNA was extracted from colontissue obtained from patients with ulcerative colitis, Crohn's diseaseor from patients with no manifestation of IBD. Real time PCR wasperformed using primers specific for STIgMA, to measure relativeexpression levels. Expression levels were 16 fold higher in a patientwith ulcerative colitis and, 5 fold higher in a patient with Crohn'sdisease, as compared to control tissue (FIG. 69A). Similarly, relativeRNA equivalents were determined in lung tissue and was found to behighest in tissue from a patient with chronic occlusive pulmonarydisease (COPD: 14 fold over normal) and was not significantly differentfrom normal in a patient with asthma (FIG. 69B).

Molecules of the Ig superfamily are well known to mediate cell surfacerecognition and cell-cell adhesion. Since STIgMA expression was high ininterstitial macrophages aligning blood vessels, STIgMA involvement inmacrophage-endothelial cell adhesion was studied. A Jurkat cell line,stably transfected with full length STIgMA-long (FIG. 70A) was loadedwith the fluorescent dye BCECF and added to the wells of a 96 wellmaxisorb plate on which a monolayer of HUVEC cells had been cultured.Adhesion was measured by the amount of fluorescence retained after 3gentle washes. Jurkat cells expressing STIgMA were more adherent toboth, control and TNFα stimulated endothelium, as compared to Jurkatcells stably transfected with a control plasmid (FIG. 70B).

Discussion

This study, for the first time, described the tissue distribution,regulation of expression and molecular characterization of a novel Igsuperfamily member STIgMA/Z39Ig and confirmed its selective expressionin tissue resident macrophages.

STIgMA expression was found on resident macrophages which had a fullydifferentiated phenotype. Its expression was increased in tissues withchronic inflammation like, rheumatoid arthritis and inflammatory boweldisease. The increase of STIgMA expression in these diseases, which wasoften characterized as Th2 type diseases, may be in line with theregulation of its expression by Th2 cytokines in vitro. Whether thisincreased expression is due to an increased presence of STIgMA positivemacrophages or an increased expression on the inflammatory macrophageshas yet to be determined.

STIgMA may mediate one of the effector functions of human macrophages,which include bacterial recognition, phagocytosis, antigen presentationand cytokine release. However, so far, no evidence was found for itsrole in any of these processes. STIgMA contained 3 tyrosine residues inits cytoplasmic domain which can be phosphorylated by tyrosine kinases.Therefore, STIgMA may act as a receptor. So far, no ligand has beenfound for STIgMA.

These results indicated a role for STIgMA in adhesion, and possiblymotility, of macrophages to the endothelial cell wall of vessels.

STIgMA expression was increased in non-microbial inflammatory diseaseslike ulcerative colitis and chronic occlusive pulmonary disease (COPD)but was downregulated in isolated macrophages upon treatment with LPS orother bacterial cell wall components like lipoteichoic acid andbacterial lipoprotein. Long term treatment, over 2 days, with LPS causedan increase in the expression of STIgMA. This could be due to anautocrine effect of IL-10 secreted by LPS-stimulated macrophages. Astriking up-regulation of STIgMA, both at the mRNA and protein levels,was observed upon treatment of monocytes or macrophages withdexamethasone. Few monocyte/macrophage surface receptors have been foundto increase in expression upon dexamethasone treatment. One example isCD163, but its induction by dexamethasone is far less dramatic. Theup-regulation of STIgMA by anti-inflammatory cytokines IL10 and TGFβ wasof considerable interest and indicates that STIgMA may mediate theanti-inflammatory role of glucocorticosteroids.

As described here, STIgMA was expressed on a subset of CD68 positivemacrophages which may represent activated macrophages. Using blockingand activating antibodies to STIgMA and STIgMA-Fc fusion protein, itsrole in macrophage effector function, adhesion and migration and itsrole in chronic inflammatory diseases has been investigated, and isdescribed in Example 25.

Only few cell surface markers were specifically expressed ondifferentiated macrophages, such as CD68 and CD163. Although CD68 wasapparently expressed on all human macrophage populations, the antigencould also be detected on other myeloid cells and also on certainnon-myeloid cells. Therefore, STIgMA represented the first cell surfaceantigen selectively expressed on a subset of interstitial maturemacrophages.

Example 25 STIGMA Fusion Proteins in Collagen-Induced Arthritis (CIA) inDBA-1J mice

This experiment aimed to compare STIgMA fusion proteins to controlmurine IgG1 in the development of disease and progression of CIA(collagen-induced arthritis, an experimental animal model system ofrheumatoid arthritis).

As discussed in Example 24, STIgMA is highly and specifically expressedon a subset of macrophages and is elevated in tissues with chronicinflammation. Murine STIgMA is highly expressed in macrophages andsynoviocytes in inflamed joints of mice with collagen-induced arthritis.In vitro studies have shown that STIgMA is involved in adhesion ofmacrophages to endothelium. STIgMA-Fc fusion protein influences thecourse of an autoimmune disease, in this case collagen-induced arthritisin mice, either by influencing the properties of tissue macrophages orby influencing immune response of other cells (e.g. T cells, B cell,epithelial cells, endothelial cells). This may result in alleviation ofinflammation, swelling and long term bone erosion in joints.

Animal Model Species: Mouse

Strain(s): DBA-1J

Supplier(s): JACKSON

Age Range: 7 to 8 week old

Pain Category: 3—These procedures cause more than minimal or transientpain and/or distress but cannot be performed using anesthetics,analgesics or tranquilizers without adversely affecting the study.

The mouse was chosen as the species to study CIA because CIA is aninflammatory polyarthritis with clinical and pathological featuressimilar to human RA (rheumatoid arthritis). This animal model has beenused by many laboratories and the histopathology of CIA resembles thoseseen in RA with synovial proliferation that progresses to pannusformation, cartilage degeneration/destruction and marginal bone erosionswith subsequent joint deformities. Also, mouse is phylogenetically thelowest mammal.

Also, there is no in vitro model available to mimic the complex,multifactorial pathogenesis of RA (Rheumatoid Arthritis).

Experimental Design

Treatment Groups:

1) mIgG1 isotype 6 mg/kg in 200 μl saline subcutaneous (SC) 3 times/wkfor 7 weeks (n=8).

2) muSTIgMA (PRO362) 4 mg/kg in 100 μl saline SC 3 times/wk for 7 weeks(n=8).

Mice were immunized interdermally with bovine CII (100 ug, Sigma, StLouis) emulsified in CFS (Difco). Mice were rechallenged with CII in IFA(Difco) 21 days later. Starting on day 24, one group of mice (n=7) wasgiven 100 ug muSTIgMA (PRO362) Fc three times per week for 6 weeks, andthe second group (n=8) received 100 ug of murine IgG1, as a control.Mice were examined daily for signs of joint inflammation and scored asfollows: 0, normal; 1, erythema and mild swelling confined to the anklejoint; 2, erythema and mild swelling extending from the ankle tometatarsal and metacarpal joints, 3 erythema and moderate swellingextending from the ankle to metatarsal or metacarpal joints. 4, erythemaand severe swelling extending from the ankle to the digits. The maximumarthritic score per paw was 4, and the maximal score per mouse was 16(FIG. 71).

All mice were immunized with 100 μg bovine collagen type II in 100 μlcomplete Freunds Adjuvant (CFA) on day 0. Collagen type II in CFA wasinjected intradermally at the base of the tail on the right side. On day21, a 2nd immunization with 100 μg bovine collagen type II in 100 μl ofincomplete Freunds adjuvant was given i.d. at the left side of the tail.Animals were checked daily (M-F) by the investigative staff. Nestletswere used as an enrichment device, and to provide extra padding for theanimals. If necessary, moistened food was provided at the bottom of thecages. Debilitated animals were sacrificed after consultation with theveterinary staff. Terminal faxitron X-Rays and microCT were taken at theend of study and joint lesion/erosion was evaluated. In addition,animals were weighed before treatment and at termination.

On day 35 and at the termination of the study, mice in Groups 1 to 8were bled for serum pK and to determine anti-collagen type II antibodytiter (100 μl orbital bleed).

On day 70 all mice were terminally bled intracardially under 3%isoflurane for a terminal hemogram, for a differential leukocyte countand for serum pK (G3) evaluation.

The mice were euthanized at day 70, post induction of arthritis. Allfour limbs were collected for radiographs, 5CT and histopathology.

Housing and Diet for Animals

Cotton pad and moistened feed was provided on the floor of the cage topromote access to food and comfort.

Drugs Used for Restraint

Isoflurane—inhalation to effect

Euthanasia Methods Exsanguination by Cardiac Puncture (Percutaneous)Under anesthesia

Isoflurane—inhalation to effect

Results

Systemic injection of the STIgMA fusion protein, muSTIgMA-Fc, into acollagen-induced arthritic mouse (animal model for rheumatoid arthritis)showed significant (see FIG. 71: p-value=0.0004) reduction in theprogression of CIA in the test group of mice that received the STIgMAfusion protein (squares) versus the control group of mice that receivedIgG1 (circles).

DEPOSIT OF MATERIAL

The following materials have been deposited with the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va.20110-2209, USA (ATCC):

Designation ATCC Dep. No. Deposit Date pRK5-based plasmid 209432 Nov. 7,1997 DNA40628-1216 DNA45416-1251 209620 Feb. 5, 1998 DNA35638-1141209265 Sep. 16, 1997 DNA77624-2515 203553 Dec. 22, 1998

These deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit. The deposit will be made available byATCC under the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the progeny of the culture of the depositto the public upon issuance of the pertinent U.S. patent or upon layingopen to the public of any U.S. or foreign patent application, whichevercomes first, and assures availability of the progeny to one determinedby the U.S. Commissioner of Patents and Trademarks to be entitledthereto according to 35 USC '122 and the Commissioner's rules pursuantthereto (including 37 CFR §1.14 with particular reference to 886 OG638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the construct deposited,since the deposited embodiment is intended as a single illustration ofcertain aspects of the invention and any constructs that arefunctionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

1. A polypeptide comprising amino acids 21 to 276 of SEQ ID NO:
 32. 2.The polypeptide of claim 1 comprising amino acids 1 to 276 of SEQ ID NO:32.
 3. An immunoadhesin comprising amino acids 21 to 276 of SEQ ID NO:32, fused to an immunoglobulin constant region sequence.
 4. Theimmunoadhesin of claim 3 wherein said constant region sequence is animmunoglobulin heavy chain constant region sequence.